B7-H1 Antibodies

ABSTRACT

The invention provides novel polypeptides useful for co-stimulating T cells, isolated nucleic acid molecules encoding them, vectors containing the nucleic acid molecules, and cells containing the vectors. Also included are methods of making and using these co-stimulatory polypeptides.

This application is a continuation of U.S. patent application Ser. No.12/347,492, filed Dec. 31, 2008, which is a continuation of U.S. patentapplication Ser. No. 09/649,108, filed Aug. 28, 2000, now abandoned,which is a continuation-in-part of U.S. patent application Ser. No.09/451,291, filed Nov. 30, 1999, now U.S. Pat. No. 6,803,192.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted in electronic andprint form. The electronic and print form of the Sequence Listing areidentical to each other pursuant to 37 CFR §1.52(e) (4), contains thefollowing file: “SEQUENCE LISTING.txt”, having a size in bytes of 30.49KB, recorded on Apr. 9, 2009. The information contained in the sequencelisting is hereby incorporated by reference in its entirety pursuant to37 CFR §1.52(e)(5).

STATEMENT OF FEDERALLY SPONSORED RESEARCH

Funding for the work described herein was provided by the federalgovernment under grant number CA079915 awarded by the National Instituteof Health. The federal government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The invention is generally in the field of immunoregulation, andspecifically T cell response regulation.

Mammalian T lymphocytes recognize antigenic peptides bound to majorhistocompatibility complex (MHC) molecules on the surface of antigenpresenting cells (APC). The antigenic peptides are generated byproteolytic degradation of protein antigens within the APC. Theinteraction of the T cells with the APC and the subsequent response ofthe T cells are qualitatively and quantitatively regulated byinteractions between cell surface receptors on the T cells with bothsoluble mediators and ligands on the surface of APC.

SUMMARY OF THE INVENTION

The invention is based on the cloning of human and mouse cDNA moleculesencoding novel homologous molecules that co-stimulate the T cellresponses of both species and on the functional characterization of thepolypeptides that the cDNA molecules encode. The human polypeptide isdesignated hB7-H1 and the mouse polypeptide mB7-H1. Text that refers toB7-H1 without specifying human versus mouse is pertinent to both formsof B7-H1. The invention features DNA molecules encoding the hB7-H1,mB7-H1 polypeptides, functional fragments of the polypeptides, andfusion proteins containing the polypeptides or functional fragments ofthe polypeptides, hB7-H1 and mB7-H1 and functional fragments of both,vectors containing the DNA molecules, and cells containing the vectors.Also included in the invention are antibodies that bind to the B7-H1polypeptides. The invention features in vitro, in vivo, and ex vivomethods of co-stimulating T cell responses, methods of screening forcompounds that inhibit or enhance T cell responses, and methods forproducing the above polypeptides and fusion proteins.

Specifically the invention features an isolated DNA including: (a) anucleic acid sequence that (i) encodes a B7-H1 polypeptide with theability to co-stimulate a T cell, and (ii) hybridizes under stringentconditions to the complement of a sequence that encodes a polypeptidewith an amino acid sequence with SEQ ID NO:1 or SEQ ID NO:3; or (b) acomplement of this nucleic acid sequence. The nucleic acid sequenceincluded in the isolated DNA will be at least 10 bp, 15 bp, 25 bp, 50bp, 75 bp, 100 bp, 125 bp, 150 bp, 175 bp, 200 bp, 250 bp, 300 bp, 350bp, 400 bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp, 700 bp, 750, bp 800bp, 850 bp, or 870 bp long. The nucleic acid sequence can encode a B7-H1polypeptide that includes an amino sequence with SEQ ID NO:1 or SEQ IDNO:3 or it can have a nucleotide sequences with SEQ ID NO:2 or SEQ IDNO:4. The nucleic acid sequence can also encode functional fragments ofthese B7-H1 polypeptides.

The invention also embodies an isolated B7-H1 polypeptide encoded by aDNA that includes a nucleic acid sequence that (i) encodes a polypeptidewith the ability to co-stimulate a T cell and (ii) hybridizes understringent conditions to the complement of a sequence that encodes apolypeptide with an amino acid sequence with SEQ ID NO:1 or SEQ ID NO:3.The B7-H1 polypeptide can include an amino sequence of amino acidresidue 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32, to aminoacid residue 290 of SEQ ID NO:1 or SEQ ID NO:3. The invention alsoencompasses B7-H1 polypeptides that include an amino acid sequence withSEQ ID NO:1 or SEQ ID NO:3, or either of these amino acid sequences butdiffering solely by one or more conservative substitutions. Thepolypeptides of the invention include fusion proteins containing a firstdomain and at least one additional domain. The first domain can be anyof the B7-H1 polypeptides described above or a functional fragment ofany of these polypeptides. The at least one additional domain can be aheterologous targeting or leader sequence, an amino acid sequence thatfacilitates purification, detection, or solubility of the fusionprotein. The second domain can be, for example, all or part of animmunoglobulin (Ig) heavy chain constant region. Also included areisolated nucleic acid molecules encoding the fusion proteins.

The invention features vectors containing any of the DNAs of theinvention and nucleic acid molecules encoding the fusion proteins of theinvention. The vectors can be expression vectors in which the nucleicacid coding sequence or molecule is operably linked to a regulatoryelement which allows expression of the nucleic acid sequence or moleculein a cell. Also included in the invention are cells (e.g., mammalian,insect, yeast, fungal, or bacterial cells) containing any of the vectorsof the invention.

Another embodiment of the invention is a method of co-stimulating a Tcell that involves contacting the T cell with any of the B7-H1polypeptides of the invention, functional fragments thereof, or fusionproteins of the invention; these 3 classes of molecule are, forconvenience, designated “B7-H1 agents”. The contacting can be byculturing any of these B7-H1 agents with the T cell in vitro.Alternatively, the T cell can be in a mammal and the contacting can be,for example, by administering any of the B7-H1 agents to the mammal oradministering a nucleic acid encoding the B7-H1 agent to the mammal. Inaddition, the method can be an ex vivo procedure that involves providinga recombinant cell which is the progeny of a cell obtained from themammal and has been transfected or transformed ex vivo with a nucleicacid encoding any of the B7-H1 agents so that the cell expresses theB7-H1 agent; and administering the cell to the mammal. In this ex vivoprocedure, the cell can be an antigen presenting cell (APC) thatexpresses the B7-H1 agent on its surface. Furthermore, prior toadministering to the mammal, the APC can be pulsed with an antigen or anantigenic peptide. In any of the above methods, the mammal can besuspected of having, for example, an immunodeficiency disease, aninflammatory condition, or an autoimmune disease. In addition, in any ofthe methods, the T cell can be a helper T cell, e.g., a T cell thathelps an effector (e.g., a cytotoxic T lymphocyte (CTL) or B cellantibody) response. An antibody response can be, for example, an IgM,IgG1, IgG2a, IgG2b, IgG3, IgG4, IgE, or IgA antibody response.Co-stimulation of a T cell by any of the B7-H1 agents can result in anincrease in the level of CD40 ligand on the surface of the T cell.

The invention includes a method of identifying a compound that inhibitsan immune response. The method involves: providing a test compound;culturing, together, the compound, one or more B7-H1 agents, a T cell,and a T cell activating stimulus; and determining whether the testcompound inhibits the response of the T cell to the stimulus, as anindication that the test compound inhibits an immune response. Theinvention also embodies a method of identifying a compound that enhancesan immune response. The method involves: providing a test compound;culturing, together, the compound, one or more of B7-H1 agents, a Tcell, and a T cell activating stimulus; and determining whether the testcompound enhances the response of the T cell to the stimulus, as anindication that the test compound enhances an immune response. In boththese methods, the stimulus can be, for example, an antibody that bindsto a T cell receptor or a CD3 polypeptide. Alternatively, the stimuluscan be an alloantigen or an antigenic peptide bound to a majorhistocompatibility complex (MHC) molecule on the surface of an antigenpresenting cell (APC). The APC can be transfected or transformed with anucleic acid encoding the B7-H1 agent and the B7-H1 agent can beexpressed on the surface of the APC.

The invention also features an antibody (e.g., a polyclonal or amonoclonal antibody) that binds to any of the B7-H1 polypeptides of theinvention, e.g., the polypeptide with SEQ ID NO:1 or SEQ ID NO:3.

The invention also features a method of producing any of the B7-H1polypeptides of the invention, functional fragments thereof, or fusionproteins of the invention. The method involves culturing a cell of theinvention and purifying the relevant B7-H1 protein from the culture.

“Polypeptide” and “protein” are used interchangeably and mean anypeptide-linked chain of amino acids, regardless of length orpost-translational modification. The invention also features B7-H1polypeptides with conservative substitutions. Conservative substitutionstypically include substitutions within the following groups: glycine andalanine; valine, isoleucine, and leucine; aspartic acid and glutamicacid; asparagine, glutamine, serine and threonine; lysine, histidine andarginine; and phenylalanine and tyrosine.

The term “isolated” polypeptide or peptide fragment as used hereinrefers to a polypeptide or a peptide fragment which either has nonaturally-occurring counterpart (e.g., a peptidomimetic), or has beenseparated or purified from components which naturally accompany it,e.g., in tissues such as pancreas, liver, spleen, ovary, testis, muscle,joint tissue, neural tissue, gastrointestinal tissue, or body fluidssuch as blood, serum, or urine. Typically, the polypeptide or peptidefragment is considered “isolated” when it is at least 70%, by dryweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, a preparation of apolypeptide (or peptide fragment thereof) of the invention is at least80%, more preferably at least 90%, and most preferably at least 99%, bydry weight, the polypeptide (or the peptide fragment thereof),respectively, of the invention. Thus, for example, a preparation ofpolypeptide x is at least 80%, more preferably at least 90%, and mostpreferably at least 99%, by dry weight, polypeptide x. Since apolypeptide that is chemically synthesized is, by its nature, separatedfrom the components that naturally accompany it, the syntheticpolypeptide or nucleic acid is “isolated.”

An isolated polypeptide (or peptide fragment) of the invention can beobtained, for example, by extraction from a natural source (e.g., fromhuman tissues or bodily fluids); by expression of a recombinant nucleicacid encoding the peptide; or by chemical synthesis. A peptide that isproduced in a cellular system different from the source from which itnaturally originates is “isolated,” because it will be separated fromcomponents which naturally accompany it. The extent of isolation orpurity can be measured by any appropriate method, e.g., columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

An “isolated DNA” means DNA free of one or both of the genes that flankthe gene containing the DNA of interest in the genome of the organism inwhich the gene containing the DNA of interest naturally occurs. The termtherefore includes a recombinant DNA incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote. It also includes a separate molecule such as: acDNA where the corresponding genomic DNA has introns and therefore adifferent sequence; a genomic fragment; a fragment produced bypolymerase chain reaction (PCR); a restriction fragment; a DNA encodinga non-naturally occurring protein, fusion protein, or fragment of agiven protein; or a nucleic acid which is a degenerate variant of anaturally occurring nucleic acid. In addition, it includes a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein. Also included is a recombinant DNA that includes aportion of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5. It will be apparentfrom the foregoing that isolated DNA does not mean a DNA present amonghundreds to millions of other DNA molecules within, for example, cDNA orgenomic DNA libraries or genomic DNA restriction digests in, forexample, a restriction digest reaction mixture or an electrophoretic gelslice.

As used herein, a polypeptide that “co-stimulates” a T cell is apolypeptide that, upon interaction with a cell-surface molecule on the Tcell, enhances the response of the T cell. The T cell response thatresults from the interaction will be greater than the response in theabsence of the polypeptide. The response of the T cell in the absence ofthe co-stimulatory polypeptide can be no response or it can be aresponse significantly lower than in the presence of the co-stimulatorypolypeptide. It is understood that the response of the T cell can be aneffector (e.g., CTL or antibody-producing B cell) response, a helperresponse providing help for one or more effector (e.g., CTL orantibody-producing B cell) responses, or a suppressive response.

As used herein, an “activating stimulus” is a molecule that delivers anactivating signal to a T cell, preferably through the antigen specific Tcell receptor (TCR). The activating stimulus can be sufficient to elicita detectable response in the T cell. Alternatively, the T cell mayrequire co-stimulation (e.g., by a B7-H1 polypeptide) in order torespond detectably to the activating stimulus. Examples of activatingstimuli include, without limitation, antibodies that bind to the TCR orto a polypeptide of the CD3 complex that is physically associated withthe TCR on the T cell surface, alloantigens, or an antigenic peptidebound to a MHC molecule.

As used herein, a “fragment” of a B7-H1 polypeptide is a fragment of thepolypeptide that is shorter than the full-length polypeptide. Generally,fragments will be five or more amino acids in length. An antigenicfragment has the ability to be recognized and bound by an antibody.

As used herein, a “functional fragment” of a B7-H1 polypeptide is afragment of the polypeptide that is shorter than the full-lengthpolypeptide and has the ability to co-stimulate a T cell. Methods ofestablishing whether a fragment of an B7-H1 molecule is functional areknown in the art. For example, fragments of interest can be made byeither recombinant, synthetic, or proteolytic digestive methods. Suchfragments can then be isolated and tested for their ability toco-stimulate T cells by procedures described herein.

As used herein, “operably linked” means incorporated into a geneticconstruct so that expression control sequences effectively controlexpression of a coding sequence of interest.

As used herein, the term “antibody” refers not only to whole antibodymolecules, but also to antigen-binding fragments, e.g., Fab, F(ab′)₂,Fv, and single chain Fv fragments. Also included are chimericantibodies.

As used herein, an antibody that “binds specifically” to an isolatedB7-H1 polypeptide encoded by a DNA that includes a nucleic acid sequencethat (i) encodes a polypeptide with the ability to co-stimulate a T celland (ii) hybridizes under stringent conditions to the complement of asequence that encodes a polypeptide with an amino acid sequence with SEQID NO:1 or SEQ ID NO:3, is an antibody that does not bind to B7-1 orB7-2 polypeptides.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention. All publications, patentapplications, patents and other references mentioned herein areincorporated by reference in their entirety. The materials, methods, andexamples disclosed herein are illustrative only and not intended to belimiting.

Other features and advantages of the invention, e.g., enhancing immuneresponses in mammalian subjects, will be apparent from the followingdescription, from the drawings and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a depiction of the nucleotide sequence of a cDNA fragment (SEQID NO: 5) that includes the coding sequence (nucleotides 72-870 of SEQID NO:5) (SEQ ID NO:2) of hB7-H1.

FIG. 2 a is a depiction of the amino acid sequence of hB7-H1 (SEQ IDNO:1). The signal peptide, Ig-V-like domain, Ig-C-like domain, andtransmembrane (“TM”) domain are indicated. Potential N-linkedglycosylation sites are indicated by *.

FIG. 2 b is a depiction of the amino acid sequences of the extracellulardomains of hB7-H1 (SEQ ID NO:10), human B7-1 (hB7-1; SEQ ID NO:11), andhuman B7-2 (hB7-2; SEQ ID NO:12) aligned for maximum homology. Identicalamino acid residues are shaded in bold and conserved residues are boxed.Conserved cysteine residues are indicated by *.

FIG. 3 is a photograph of a Northern blot showing expression of hB7-H1mRNA in various human tissues.

FIG. 4 is a series of two-dimensional fluorescence flow cytometryhistograms showing cell surface expression of hB7-H1 on resting andactivated CD3+ T cells, CD19+ B cells, and CD 14+ monocytes.

FIG. 5 a is a series of fluorescence flow cytometry histograms showingbinding of CTLA-4Ig, ICOSIg, and antibody specific for hB7-H1 to 293cells transfected with either a control vector (“Mock/293 cells”) or avector containing a cDNA insert encoding hB7-H1 (“hB7-H1/293 cells”), orRaji cells.

FIG. 5 b is a series of fluorescence flow cytometry histograms showingthe binding of hB7-H1Ig and antibody to CD28 to Jurkat cells.

FIG. 6 a is a line graph showing the ability of immobilized hB7-H1Ig toco-stimulate the proliferative response of human T cells to immobilizedantibody specific for human CD3.

FIG. 6 b is a line graph showing the ability of soluble hB7-H1Ig toco-stimulate the proliferative response of human T cells to irradiatedallogeneic PBMC.

FIGS. 7 a-7 d are a series of line graphs showing the ability ofhB7-H1Ig, human B7-1Ig, or antibody specific for human CD28 toco-stimulate the production of interleukin-(IL-)10 (FIG. 7 a),interferon-γ (IFN-γ) (FIG. 7 b), IL-2 (FIG. 7 c), or IL-4 (FIG. 7 d) byhuman T cells responding to immobilized antibody specific for CD3. FIG.7 e is a line graph showing the ability of various concentrations ofhB7-H1Ig to co-stimulate the production of IL-2 by human T cellsresponding to immobilized antibody specific for CD3.

FIG. 8 a is a bar graph showing the ability of antibody specific forhuman IL-2 to inhibit the proliferation of human T cells induced byantibody specific for human CD3 and co-stimulated by COS cellstransfected with and expressing either hB7-H1 or human-B7-1.

FIG. 8 b is a bar graph showing the ability of antibody specific forhuman IL-2 to inhibit the production of IL-10 by human T cellsstimulated by immobilized antibody specific for human CD3 andco-stimulated by either hB7-H1Ig or B7-1Ig.

FIG. 9 a is a series of two-dimensional fluorescence flow cytometryprofiles showing the relative proportion of T cells in early (annexinV-positive, propidium iodide (PI)-negative) and late (annexinV-positive, PI-positive) apoptosis following activation by immobilizedantibody specific for human CD3 and co-stimulation with either controlIg or hB7-H1Ig.

FIG. 9 b is series of fluorescence flow cytometry profiles showingexpression of Fas and FasL on human T cells following activation byimmobilized antibody specific for human CD3 and co-stimulation witheither control Ig or hB7-H1Ig.

FIG. 10 is a depiction of the nucleotide sequence of cDNA encodingmB7-H1 (SEQ ID NO:4).

FIG. 11 is a depiction of the amino acid sequence of mB7-H1 (SEQ IDNO:3).

FIG. 12 a is a depiction of the amino acid sequence of mB7-H1 (SEQ IDNO:3) aligned with the amino acid sequence of hB7-H1 (SEQ ID NO:1). Thesignal peptide, IgV-like domain, IgC-like domain, transmembrane (“TM”)domain, and cytoplasmic domain (“cytoplasmic”) of in B7-H1 areindicated.

FIG. 12 b is a depiction of the amino acid sequences of mB7-H1, mouseB7-1 (mB7-1; SEQ ID NO:13), mouse B7-2 (mB7-2: SEQ ID NO:14), andmB7h/B7KF-1 (SEQ ID NO:15) aligned for maximum homology. Identical aminoacid residues are shaded and conserved amino acid residues are boxed.Conserved cysteine residues are indicated by *.

FIG. 13 is a series of fluorescence flow cytometry histograms showingrelative levels of cell surface expression of mB7-H1 on resting andactivated mouse CD3+ T cells, mouse B220+ B cells, and Mac-1+ mousemacrophages.

FIG. 14 a is a line graph showing the ability of various concentrationsof immobilized mB7-H1Ig or immobilized control Ig to co-stimulate the invitro proliferative response of mouse T cells to a suboptimal dose ofimmobilized antibody specific for mouse CD3.

FIG. 14 b is a pair of line graphs showing the ability of immobilizedmB7-H1Ig, immobilized control Ig, or soluble antibody specific for mouseCD28 to co-stimulate the in vitro proliferative response of wild type(“wt”) C57BL/6 mouse (left graph) or CD28-deficient (“CD28^(−/−)”)C57BL/6 mouse (right graph) T cells to two suboptimal doses ofimmobilized antibody specific for mouse CD3.

FIG. 14 c is a bar graph showing the ability of immobilized mB7-H1Ig orimmobilized control Ig to co-stimulate the in vitro proliferativeresponse of purified CD4+ or CD8+ mouse T cells to a suboptimal dose ofimmobilized antibody specific for mouse CD3.

FIG. 15 a is a series of line graphs showing the ability of immobilizedmB7-H1Ig, immobilized control Ig, or soluble antibody specific for mouseCD28 (“Anti-CD28”) to co-stimulate the in vitro production (on days one,two, and three after initiation of the cultures) of various cytokines byC57BL/6 mouse T cells in response to immobilized antibody specific formouse CD3.

FIG. 15 b is a line graph showing the effect of immobilized mB7-H1Ig onthe in vitro production (18, 24, 36, and 48 hours after initiation ofthe cultures) of IL-2 by C57BL/6 mouse T cells responding to immobilizedantibody specific for mouse CD3 and co-stimulated by soluble antibodyspecific for mouse CD28 (“Anti-CD28”).

FIG. 16 a and FIG. 16 b are fluorescence flow cytometry histogramsshowing lack of surface expression of murine B7-1 (FIG. 16 a) and mB7-H1(FIG. 16 b) on P815 cells transfected with a control expression vector(“mock.P815”).

FIG. 17 a and FIG. 17 b are fluorescence flow cytometry histogramsshowing surface expression of murine B7-1 (FIG. 17 a) and lack ofsurface expression of mB7-1 (FIG. 17 b) on P815 cells transfected withan expression vector containing a nucleic acid sequence encoding murineB7-1 (“mB7-1⁺ P815”).

FIG. 18 a and FIG. 18 b are fluorescence flow cytometry histogramsshowing lack of surface expression of mB7-1 (FIG. 18 a) and showingsurface expression of mB7-H1 (FIG. 18 b) on P815 cells transfected withan expression vector containing a nucleic acid sequence encoding mB7-H1(“mB7-H1⁺ P815”).

FIG. 19 a and FIG. 19 b are line graphs showing the growth rate of P815tumors in DBA/2 mice injected subcutaneously with P815 cells transfectedwith a control expression vector (FIG. 19 a) or an expression vectorcontaining a nucleic acid sequence encoding mB7-H1 (FIG. 19 b).

FIG. 20 a is a pair of line graphs showing the ability of controlmock-transfected P815 cells (“mock P815”), P815 cells transfected withan expression vector containing a cDNA sequence encoding mB7-H1(“mB7-H1⁺ P815”), or P815 cells transfected with an expression vectorcontaining a cDNA sequence encoding mB7-1 (“mB7-1⁺ P815”) to activateallospecific C57BL/6 mouse CTL in vitro. The effector cell populationswere tested for cytotoxic activity (“% Lysis”) at various effector totarget cell (E/T) ratios against wild type P815 (left graph) and controlEL4 (right graph) target cells.

FIG. 20 b is a pair of line graphs showing the ability of controlmock-transfected P815 cells (“mock. P815”), P815 cells transfected withan expression vector containing a cDNA sequence encoding mB7-H1(“mB7-H1⁺ P815”), or P815 cells transfected with an expression vectorcontaining a cDNA sequence encoding mB7-1 (“mB7-1⁺ P815”) to activatetumor-specific DBA/2 mouse CTL in vivo. The effector cell populationswere tested for cytotoxic activity (“% Lysis”) at various E/T ratiosagainst wild type P815 (left graph) and control L1210 (right graph)target cells.

FIG. 21 is a pair of line graphs showing the in vitro proliferativeresponses to various concentrations of keyhole limpet hemacyanin (KLH)of draining lymph node (left graph) or spleen (right graph) T cells fromC57BL/6 immunized intraperitoneally (i.p.) with trinitrophenol (TNP)conjugated KLH (TNP-KLH) in incomplete Freund's adjuvant andsubsequently injected i.p. with mB7-H1Ig or control Ig.

FIG. 22 is a series of bar graphs showing the relative levels (asmeasured by ELISA) of IgG1, IgG2a, IgG2b, IgG3, or IgM antibodiesspecific for TNP in the sera of C57BL/6 mice injected i.p. with TNP-KLHin phosphate buffered saline and either control Ig, mB7-H1Ig, ormB7-1Ig.

FIG. 23 a is a series of fluorescence flow cytometry histograms showingthe relative levels of cell surface expression of CD40 ligand (CD40L)(as measured by the binding of an antibody specific for mouse CD40L(“anti-CD40L”)) on purified mouse CD4+ T cells (at 4 and 24 hours afterinitiation of the cultures) activated by immobilized antibody specificfor mouse CD3 (“anti-CD3”) coated onto the bottoms of tissue culturewells at a concentration of 200 ng/ml and co-stimulated by immobilizedcontrol Ig, immobilized mB7-H1Ig, or soluble antibody specific for mouseCD28 (“anti-CD28”).

FIG. 23 b is a series of fluorescence flow cytometry histograms showingthe relative levels of cell surface expression of CD40 ligand (CD40L)(as measured by the binding of an antibody specific for mouse CD40L(“anti-CD40L”)) on purified mouse CD4+ T cells (at 4 and 24 hours afterinitiation of the cultures) activated by immobilized antibody specificfor mouse CD3 (“anti-CD3”) coated onto the bottoms of tissue culturewells at a concentration of 1,000 ng/ml and co-stimulated by immobilizedcontrol Ig, immobilized mB7-H1Ig, or soluble antibody specific for mouseCD28 (“anti-CD28”).

DETAILED DESCRIPTION

Using PCR primers with sequences derived from an expressed sequence tag(EST) that had significant homology to human B7-1 and B7-2 and a humancDNA library as a source of template, cDNA sequences corresponding toregions of a transcript 5′ and 3′ of the EST were identified. A cDNAmolecule (SEQ ID NO:5) that included a open reading frame (orf) (SEQ IDNO:2) encoding a novel B7-related molecule was then generated using PCRprimers with sequences derived from the 3′ and 5′ ends and cloned.

Translation of the cDNA sequence indicated that the polypeptide (SEQ IDNO:1) that it encoded (hB7-H1) is a type I transmembrane protein of 290amino acids containing an immunoglobulin (Ig) V-like domain, Ig C-likedomain, a transmembrane domain and a cytoplasmic domain of 30 aminoacids. Northern blot analysis showed strong expression of the geneencoding hB7-H1 in heart, skeletal muscle, placenta, and lung, and weakexpression in thymus, spleen, kidney, and liver. Expression wasundetectable in brain, colon, small intestine, and peripheral bloodmononuclear cells (PBMC).

Using an antiserum produced by immunization of mice with a recombinantlyproduced fusion protein that included the hB7-H1 protein, expression byfluorescence flow cytometry indicated negligible expression on resting Tand B cells. On the other hand, about 16% of CD14+ monocytesconstitutively expressed the molecule on their surface. Activation of Tcells increased expression such that about 30% expressed cell-surfacehB7-H1. Activation resulted in about 90% of monocytes expressing hB7-1H,but only about 6% of B cells expressed it after activation.

Transfection of 293 cells resulted in an hB7-H1 expressing cell line(hB7-H1/293) which was used for binding experiments. These experimentsand others with a CD28 expressing cell line indicated that neitherCTLA4, ICOS, nor CD28 were receptors for hB7-H1.

In vitro experiments with isolated human T cells and thehB7-H1-containing fusion protein indicated that hB7-H1 had no directactivity on T cells, it enhanced (“co-stimulated”) T cell proliferativeresponses induced by both antibody specific for human CD3 and MHCalloantigens. This co-stimulatory activity was significantly more potentwhen the hB7-H1 was immobilized in the plastic tissue culture wells usedfor the cultures than when it as in solution. Similar experimentsindicated that hB7-H1 had a dramatic and selective enhancing effect onthe production of interleukin (IL)-10 induced by T cell activation.Moreover this IL-10 enhancing activity appeared to be dependent on atleast low amounts of IL-2. Analysis of T cells activated by anti-CD3antibody and hB7-H1Ig indicated that hB7-H1 enhances apoptosis andexpression of Fas and FasL

In addition, using a strategy similar to that used to clone hB7-H1 cDNA,a cDNA molecule containing an orf encoding mouse B7-H1 (mB7-H1) wascloned, the nucleotide sequence of the orf (SEQ ID NO:4) was obtained,and the amino acid sequence of the encoded sequence (SEQ ID NO:3) wasderived. mB7-H1 is exactly the same length (290 amino acids) and has thesame domain structure as hB7-H1. Moreover, mB7-H1 has a similar tissuedistribution to hB7-H1. mB7-H1 co-stimulated the response of mouse Tcells with its effect being more potent on CD4+ than on CD8+ T cells. Inaddition, like hB7-H1, mB7-H1 co-stimulates the production of highlevels of IL-10 by T cells. mB7-H1 also enhanced the production of bothinterferon-γ (IFN-γ) and granulocyte macrophage-colony stimulatingfactor (GM-CSF) by T cells. While mB7-H1 showed no significant abilityto enhance CTL responses, it did greatly increase antibody responsesand, in particular, IgG2a antibody responses. Finally, co-stimulation ofT cells with mB7-H1 caused an increase in the level of CD40 ligand(CD40L) on the surface of the T cells.

B7-H1 can be useful as an augmenter of immune responses (e.g., helper Tcell and antibody responses) both in vivo and in vitro. Furthermore, inlight of (a) its ability to selectively enhance IL-10 production, (b)its ability to enhance apoptosis, and (c) is expression in placenta andlung, both organs normally protected from unneeded cellular-mediatedimmune and inflammatory responses, B7-H1 can be useful in controllingpathologic cell-mediated conditions (e.g., those induced by infectiousagents such Mycobacterium tuberculosis or M. leprae) or other pathologiccell-mediated responses such as those involved in autoimmune diseases(e.g., rheumatoid arthritis (RA), multiple sclerosis (MS), orinsulin-dependent diabetes mellitus (IDDM)).

B7-H1 Nucleic Acid Molecules

The B7-H1 nucleic acid molecules of the invention can be cDNA, genomicDNA, synthetic DNA, or RNA, and can be double-stranded orsingle-stranded (i.e., either a sense or an antisense strand). Segmentsof these molecules are also considered within the scope of theinvention, and can be produced by, for example, the polymerase chainreaction (PCR) or generated by treatment with one or more restrictionendonucleases. A ribonucleic acid (RNA) molecule can be produced by invitro transcription. Preferably, the nucleic acid molecules encodepolypeptides that, regardless of length, are soluble under normalphysiological conditions the membrane forms would not be soluble.

The nucleic acid molecules of the invention can contain naturallyoccurring sequences, or sequences that differ from those that occurnaturally, but, due to the degeneracy of the genetic code, encode thesame polypeptide (for example, the polypeptides with SEQ ID NOS:1 and3). In addition, these nucleic acid molecules are not limited to codingsequences, e.g., they can include some or all of the non-codingsequences that lie upstream or downstream from a coding sequence. Theyinclude, for example, the nucleic acid molecule with SEQ ID NO:5.

The nucleic acid molecules of the invention can be synthesized (forexample, by phosphoramidite-based synthesis) or obtained from abiological cell, such as the cell of a mammal. Thus, the nucleic acidscan be those of a human, non-human primate (e.g., monkey) mouse, rat,guinea pig, cow, sheep, horse, pig, rabbit, dog, or cat.

In addition, the isolated nucleic acid molecules of the inventionencompass segments that are not found as such in the natal state. Thus,the invention encompasses recombinant nucleic acid molecules, (forexample, isolated nucleic acid molecules encoding hB7-H1 or mB7-H1)incorporated into a vector (for example, a plasmid or viral vector) orinto the genome of a heterologous cell (or the genome of a homologouscell, at a position other than the natural chromosomal location).Recombinant nucleic acid molecules and uses therefor are discussedfurther below.

Certain nucleic acid molecules of the invention are antisense moleculesor are transcribed into antisense molecules. These can be used, forexample, to down-regulate translation of B7-H1 mRNA within a cell.

Techniques associated with detection or regulation of genes are wellknown to skilled artisans and such techniques can be used to diagnoseand/or treat disorders associated with aberrant B7-H1 expression.Nucleic acid molecules of the invention are discussed further below inthe context of their therapeutic utility.

A B7-H1 family gene or protein can be identified based on its similarityto the relevant B7-H1 gene or protein, respectively. For example, theidentification can be based on sequence identity. The invention featuresisolated nucleic acid molecules which are at least 50% (or 55%, 65%,75%, 85%, 95%, or 98%) identical to: (a) a nucleic acid molecule thatencodes the polypeptide of SEQ ID NO:1 or 3; (b) the nucleotide sequenceof SEQ ID NO:2 or 4; or (c) a nucleic acid molecule which includes asegment of at least 30 (e.g., at least 50, 60, 100, 125, 150, 175, 200,250, 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, or865) nucleotides of SEQ ED NO:2 or SEQ ID NO:4.

The determination of percent identity between two sequences isaccomplished using the mathematical algorithm of Karlin and Altschul,Proc. Natl. Acad. Sci. USA 90, 5873-5877, 1993. Such an algorithm isincorporated into the BLASTN and BLASTP programs of Altschul et al.(1990) J. Mol. Biol. 215, 403-410. BLAST nucleotide searches areperformed with the BLASTN program, score=100, wordlength=12 to obtainnucleotide sequences homologous to B7-H1-encoding nucleic acids. BLASTprotein searches are performed with the BLASTP program, score=50,wordlength=3 to obtain amino acid sequences homologous to B7-H1. Toobtain gapped alignments for comparative purposes, Gapped BLAST isutilized as described in Altschul et al. (1997) Nucleic Acids Res. 25,3389-3402. When utilizing BLAST and Gapped BLAST programs, the defaultparameters of the respective programs (e.g., XBLAST and NBLAST) are used(See http://www.ncbi.nlm.nih.gov).

Hybridization can also be used as a measure of homology between twonucleic acid sequences. A B7-H1-encoding nucleic acid sequence, or aportion thereof, can be used as hybridization probe according tostandard hybridization techniques. The hybridization of a B7-H1 probe toDNA from a test source (e.g., a mammalian cell) is an indication of thepresence of B7-H1 DNA in the test source. Hybridization conditions areknown to those skilled in the art and can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6, 1991.Moderate hybridization conditions are defined as equivalent tohybridization in 2× sodium chloride/sodium citrate (SSC) at 30° C.,followed by one or more washes in 1×SSC, 0.1% SDS at 50-60° C. Highlystringent conditions are defined as equivalent to hybridization in 6×sodium chloride/sodium citrate (SSC) at 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65° C.

The invention also encompasses: (a) vectors that contain any of theforegoing B7-H1-related coding sequences and/or their complements (thatis, “antisense” sequence); (b) expression vectors that contain any ofthe foregoing B7-H1-related coding sequences operatively associated withany transcriptional/translational regulatory elements (examples of whichare given below) necessary to direct expression of the coding sequences;(c) expression vectors containing, in addition to sequences encoding aB7-H1 polypeptide, nucleic acid sequences that are unrelated to nucleicacid sequences encoding B7-H1, such as molecules encoding a reporter,marker, or a signal peptide, e.g., fused to B7-H1; and (d) geneticallyengineered host cells that contain any of the foregoing expressionvectors and thereby express the nucleic acid molecules of the invention.

Recombinant nucleic acid molecules can contain a sequence encodinghB7-H1 or mB7-H1, or B7-H1 having an heterologous signal sequence. Thefull length B7-H1 polypeptide, a domain of B7-H1, or a fragment thereofmay be fused to additional polypeptides, as described below. Similarly,the nucleic acid molecules of the invention can encode the mature formof B7-H1 or a form that includes an exogenous polypeptide whichfacilitates secretion.

The transcriptional/translational regulatory elements referred to aboveand which are further described below, include, but are not limited to,inducible and non-inducible promoters, enhancers, operators and otherelements, which are known to those skilled in the art, and which driveor otherwise regulate gene expression. Such regulatory elements includebut are not limited to the cytomegalovirus hCMV immediate early gene,the early or late promoters of SV40 adenovirus, the lac system, the trpsystem, the TAC system, the TRC system, the major operator and promoterregions of phage A, the control regions of fd coat protein, the promoterfor 3-phosphoglycerate kinase, the promoters of acid phosphatase, andthe promoters of the yeast α-mating factors.

Similarly, the nucleic acid can form part of a hybrid gene encodingadditional polypeptide sequences, for example, sequences that functionas a marker or reporter. Examples of marker or reporter genes includeβ-lactamase, chloramphenicol acetyltransferase (CAT), adenosinedeaminase (ADA), aminoglycoside phosphotransferase (neo^(r), G418^(r)),dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH),thymidine kinase (TK), lacZ (encoding β-galactosidase), and xanthineguanine phosphoribosyltransferase (XGPRT). As with many of the standardprocedures associated with the practice of the invention, skilledartisans will be aware of additional useful reagents, for example,additional sequences that can serve the function of a marker orreporter. Generally, the hybrid polypeptide will include a first portionand a second portion; the first portion being a B7-H1 polypeptide andthe second portion being, for example, the reporter described above oran Ig constant region or part of an Ig constant region, e.g., the CH2and CH3 domains of IgG2a heavy chain.

The expression systems that may be used for purposes of the inventioninclude, but are not limited to, microorganisms such as bacteria (forexample, E. coli and B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA, or cosmid DNA expression vectorscontaining the nucleic acid molecules of the invention; yeast (forexample, Saccharomyces and Pichia) transformed with recombinant yeastexpression vectors containing the nucleic acid molecules of theinvention (preferably containing the nucleic acid sequence encodingB7-H1 (contained within SEQ ID NOS:1 or 3); insect cell systems infectedwith recombinant virus expression vectors (for example, baculovirus)containing the nucleic acid molecules of the invention; plant cellsystems infected with recombinant virus expression vectors (for example,cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) ortransformed with recombinant plasmid expression vectors (for example, Tiplasmid) containing B7-H1 nucleotide sequences; or mammalian cellsystems (for example, COS, CHO, BHK, 293, VERO, HeLa, MDCK, WI38, andNIH 3T3 cells) harboring recombinant expression constructs containingpromoters derived from the genome of mammalian cells (for example, themetallothionein promoter) or from mammalian viruses (for example, theadenovirus late promoter and the vaccinia virus 7.5K promoter). Alsouseful as host cells are primary or secondary cells obtained directlyfrom a mammal, transfected with a plasmid vector or infected with aviral vector.

Polypeptides and Polypeptide Fragments

The polypeptides of the invention include hB7-H1, mB7-H1, and functionalfragments of these polypeptides. The polypeptides embraced by theinvention also include fusion proteins which contain either full-lengthB7-H1 or a functional fragment of it used to unrelated amino acidsequence. The unrelated sequences can be additional functional domainsor signal peptides. Signal peptides are described in greater detail andexemplified below. The polypeptides can be any of those described abovebut with one or more (e.g., one, two, three, four, five, six, seven,eight, nine, 10, 12, 14, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100or more) conservative substitutions.

The polypeptides can be purified from natural sources (e.g., blood,serum plasma, tissues or cells such as T cells or any cell thatnaturally produces B7-H1). Smaller peptides (less than 50 amino acidslong) can also be conveniently synthesized by standard chemical means.In addition, both polypeptides and peptides can be produced by standardin vitro recombinant DNA techniques and in vivo recombination/geneticrecombination (e.g., transgenesis), using the nucleotide sequencesencoding the appropriate polypeptides or peptides. Methods well known tothose skilled in the art can be used to construct expression vectorscontaining relevant coding sequences and appropriatetranscriptional/translational control signals. See for example, thetechniques described in Sambrook et al., Molecular Cloning: A LaboratoryManual (2nd Ed.) [Cold Spring Harbor Laboratory, N.Y., 1989], andAusubel et al., Current Protocols in Molecular Biology, [GreenPublishing Associates and Wiley Interscience, N.Y., 1989].

Polypeptides and fragments of the invention also include those describedabove, but modified for in vivo use by the addition, at the amino-and/or carboxyl-terminal ends, of a blocking agent to facilitatesurvival of the relevant polypeptide in vivo. This can be useful inthose situations in which the peptide termini tend to be degraded byproteases prior to cellular uptake. Such blocking agents can include,without limitation, additional related or unrelated peptide sequencesthat can be attached to the amino and/or carboxyl terminal residues ofthe peptide to be administered. This can be done either chemicallyduring the synthesis of the peptide or by recombinant DNA technology bymethods familiar to artisans of average skill.

Alternatively, blocking agents such as pyroglutamic acid or othermolecules known in the art can be attached to the amino and/or carboxylterminal residues, or the amino group at the amino terminus or carboxylgroup at the carboxyl terminus can be replaced with a different moiety.Likewise, the peptides can be covalently or noncovalently coupled topharmaceutically acceptable “carrier” proteins prior to administration.

Also of interest are peptidomimetic compounds that are designed basedupon the amino acid sequences of the functional peptide fragments.Peptidomimetic compounds are synthetic compounds having athree-dimensional conformation (i.e., a “peptide motif”) that issubstantially the same as the three-dimensional conformation of aselected peptide. The peptide motif provides the peptidomimetic compoundwith the ability to co-stimulate T cells in a manner qualitativelyidentical to that of the B7-H1 functional peptide fragment from whichthe peptidomimetic was derived. Peptidomimetic compounds can haveadditional characteristics that enhance their therapeutic utility, suchas increased cell permeability and prolonged biological half-life.

The peptidomimetics typically have a backbone that is partially orcompletely non-peptide, but with side groups that are identical to theside groups of the amino acid residues that occur in the peptide onwhich the peptidomimetic is based. Several types of chemical bonds,e.g., ester, thioester, thioamide, retroamide, reduced carbonyl,dimethylene and ketomethylene bonds, are known in the art to begenerally useful substitutes for peptide bonds in the construction ofprotease-resistant peptidomimetics.

Methods of Co-Stimulating a T Cell

The methods of the invention involve contacting a T cell with a B7-H1polypeptide of the invention, or a functional fragment thereof, in orderto co-stimulate the T cell. Such polypeptides or functional fragmentscan have amino acid sequences identical to wild-type sequences or theycan contain one or more (e.g., one, two, three, four, five, six, seven,eight, nine, 10, 12, 14, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100or more) conservative substitutions. The contacting can occur before,during, or after activation of the T cell. Contacting of the T cell withthe B7-H1 polypeptide will preferably be at substantially the same timeas activation. Activation can be, for example, by exposing the T cell toan antibody that binds to the TCR or one of the polypeptides of the CD3complex that is physically associated with the TCR. Alternatively, the Tcell can be exposed to either an alloantigen (e.g., a MHC alloantigen)on, for example, an antigen presenting cell (APC) (e.g., a dendriticcell, a macrophage, a monocyte, or a B cell) or an antigenic peptideproduced by processing of a protein antigen by any of the above APC andpresented to the T cell by MHC molecules on the surface of the APC. TheT cell can be a CD4+ T cell or a CD8+ T cell. The B7-H1 molecule can beadded to the solution containing the cells, or it can be expressed onthe surface of an APC, e.g., an APC presenting an alloantigen or anantigen peptide bound to an MHC molecule. Alternatively, if theactivation is in vitro, the B7-H1 molecule can be bound to the floor ofa the relevant culture vessel, e.g. a well of a plastic microtiterplate.

The methods can be performed in vitro, in vivo, or ex vivo. In vitroapplication of B7-H1 can be useful, for example, in basic scientificstudies of immune mechanisms or for production of activated T cells foruse in either studies on T cell function or, for example, passiveimmunotherapy. Furthermore, B7-H1 could be added to in vitro assays(e.g., in T cell proliferation assays) designed to test for immunity toan antigen of interest in a subject from which the T cells wereobtained. Addition of B7-H1 to such assays would be expected to resultin a more potent, and therefore more readily detectable, in vitroresponse. However, the methods of the invention will preferably be invivo or ex vivo (see below).

The B7-H1 proteins and variants thereof are generally useful as immuneresponse-stimulating therapeutics. For example, the polypeptides of theinvention can be used for treatment of disease conditions characterizedby immunosuppression: e.g., cancer, AIDS or AIDS-related complex, othervirally or environmentally-induced conditions, and certain congenitalimmune deficiencies. The compounds may also be employed to increaseimmune function that has been impaired by the use of radiotherapy ofimmunosuppressive drugs such as certain chemotherapeutic agents, andtherefore are particularly useful when given in conjunction with suchdrugs or radiotherapy. In addition, in view of the ability of B7-H1 toco-stimulate the production of especially high levels of IL-10, B7-H1molecules can be used to treat conditions involving cellular immuneresponses, e.g., inflamatory conditions, e.g., those induced byinfectious agents such Mycobacterium tuberculosis or M. leprae, or otherpathologic cell-mediated responses such as those involved in autoimmunediseases (e.g., rheumatoid arthritis (RA), multiple sclerosis (MS), orinsulin-dependent diabetes mellitus (IDDM)).

These methods of the invention can be applied to a wide range ofspecies, e.g., humans, non-human primates, horses, cattle, pigs, sheep,goats, dogs, cats, rabbits, guinea pigs, hamsters, rats, and mice.

In Vivo Approaches

In one in vivo approach, the B7-H1 polypeptide (or a functional fragmentthereof) itself is administered to the subject. Generally, the compoundsof the invention will be suspended in a pharmaceutically-acceptablecarrier (e.g., physiological saline) and administered orally or byintravenous infusion, or injected subcutaneously, intramuscularly,intraperitoneally, intrarectally, intravaginally, intranasally,intragastrically, intratracheally, or intrapulmonarily. They arepreferably delivered directly to an appropriate lymphoid tissue (e.g.spleen, lymph node, or mucosal-associated lymphoid tissue (MALT)). Thedosage required depends on the choice of the route of administration,the nature of the formulation, the nature of the patient's illness, thesubject's size, weight, surface area, age, and sex, other drugs beingadministered, and the judgment of the attending physician. Suitabledosages are in the range of 0.01-100.0 μg/kg. Wide variations in theneeded dosage are to be expected in view of the variety of polypeptidesand fragments available and the differing efficiencies of various routesof administration. For example, oral administration would be expected torequire higher dosages than administration by i.v. injection. Variationsin these dosage levels can be adjusted using standard empirical routinesfor optimization as is well understood in the art. Administrations canbe single or multiple (e.g., 2- or 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-,150-, or more fold). Encapsulation of the polypeptide in a suitabledelivery vehicle (e.g., polymeric microparticles or implantable devices)may increase the efficiency of delivery, particularly for oral delivery.

Alternatively, a polynucleotide containing a nucleic acid sequenceencoding the B7-H1 polypeptide or functional fragment can be deliveredto an appropriate cell of the animal. Expression of the coding sequencewill preferably be directed to lymphoid tissue of the subject by, forexample, delivery of the polynucleotide to the lymphoid tissue. This canbe achieved by, for example, the use of a polymeric, biodegradablemicroparticle or microcapsule delivery vehicle, sized to optimizephagocytosis by phagocytic cells such as macrophages. For example, PLGA(poly-lacto-co-glycolide) microparticles approximately 1-10 μm indiameter can be used. The polynucleotide is encapsulated in thesemicroparticles, which are taken up by macrophages and graduallybiodegraded within the cell, thereby releasing the polynucleotide. Oncereleased, the DNA is expressed within the cell. A second type ofmicroparticle is intended not to be taken up directly by cells, butrather to serve primarily as a slow-release reservoir of nucleic acidthat is taken up by cells only upon release from the micro-particlethrough biodegradation. These polymeric particles should therefore belarge enough to preclude phagocytosis (i.e., larger than 5 μm andpreferably larger than 20 μm.

Another way to achieve uptake of the nucleic acid is using liposomes,prepared by standard methods. The vectors can be incorporated alone intothese delivery vehicles or co-incorporated with tissue-specificantibodies. Alternatively, one can prepare a molecular conjugatecomposed of a plasmid or other vector attached to poly-L-lysine byelectrostatic or covalent forces. Poly-L-lysine binds to a ligand thatcan bind to a receptor on target cells [Cristiano et al. (1995), J. Mol.Med. 73, 479]. Alternatively, lymphoid tissue specific targeting can beachieved by the use of lymphoid tissue-specific transcriptionalregulatory elements (TRE) such as a B lymphocyte, T lymphocyte, ordendritic cell specific TRE. Lymphoid tissue specific TRE are known[Thompson et al. (1992), Mol. Cell. Biol. 12, 1043-1053; Todd et al.(1993), J. Exp. Med. 177, 1663-1674; Penix et al. (1993), J. Exp. Med.178, 1483-1496]. Delivery of “naked DNA” (i.e., without a deliveryvehicle) to an intramuscular, intradermal, or subcutaneous site, isanother means to achieve in vivo expression.

In the relevant polynucleotides (e.g., expression vectors) the nucleicacid sequence encoding the B7-H1 polypeptide or functional fragment ofinterest with an initiator methionine and optionally a targetingsequence is operatively linked to a promoter or enhancer-promotercombination.

Short amino acid sequences can act as signals to direct proteins tospecific intracellular compartments. For example, hydrophobic signalpeptides (e.g., MAISGVPVLGFFIIAVLMSAQESWA (SEQ ID NO:6)) are found atthe amino terminus of proteins destined for the ER. While the sequenceKFERQ (SEQ ID NO:7) (and other closely related sequences) is known totarget intracellular polypeptides to lysosomes, other sequences (e.g.,MDDQRDLISNNEQLP (SEQ ID NO:8) direct polypeptides to endosomes. Inaddition, the peptide sequence KDEL (SEQ ID NO:9) has been shown to actas a retention signal for the ER. Each of these signal peptides, or acombination thereof, can be used to traffic the B7-H1 polypeptides orfunctional fragments of the invention as desired. DNAs encoding theB7-H1 polypeptides or functional fragments containing targeting signalswill be generated by PCR or other standard genetic engineering orsynthetic techniques.

A promoter is a TRE composed of a region of a DNA molecule, typicallywithin 100 basepairs upstream of the point at which transcriptionstarts. Enhancers provide expression specificity in terms of time,location, and level. Unlike a promoter, an enhancer can function whenlocated at variable distances from the transcription site, provided apromoter is present. An enhancer can also be located downstream of thetranscription initiation site. To bring a coding sequence under thecontrol of a promoter, it is necessary to position the translationinitiation site of the translational reading frame of the peptide orpolypeptide between one and about fifty nucleotides downstream (3′) ofthe promoter. The coding sequence of the expression vector isoperatively linked to a transcription terminating region.

Suitable expression vectors include plasmids and viral vectors such asherpes viruses, retroviruses, vaccinia viruses, attenuated vacciniaviruses, canary pox viruses, adenoviruses and adeno-associated viruses,among others.

Polynucleotides can be administered in a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers are biologicallycompatible vehicles which are suitable for administration to a human,e.g., physiological saline. A therapeutically effective amount is anamount of the polynucleotide which is capable of producing a medicallydesirable result (e.g., an enhanced T cell response) in a treatedanimal. As is well known in the medical arts, the dosage for any onepatient depends upon many factors, including the patient's size, bodysurface area, age, the particular compound to be administered, sex, timeand route of administration, general health, and other drugs beingadministered concurrently. Dosages will vary, but a preferred dosage foradministration of polynucleotide is from approximately 10⁶ to 10¹²copies of the polynucleotide molecule. This dose can be repeatedlyadministered, as needed. Routes of administration can be any of thoselisted above.

Ex Vivo Approaches

Peripheral blood mononuclear cells (PBMC) can be withdrawn from thepatient or a suitable donor and exposed ex vivo to an activatingstimulus (see above) and a B7-H1 polypeptide or polypeptide fragment(whether in soluble form or attached to a sold support by standardmethodologies). The PBMC containing highly activated T cells are thenintroduced into the same or a different patient.

An alternative ex vivo strategy can involve transfecting or transducingcells obtained from the subject with a polynucleotide encoding an B7-H1polypeptide or functional fragment-encoding nucleic acid sequencesdescribed above. The transfected or transduced cells are then returnedto the subject. While such cells would preferably be hemopoietic cells(e.g., bone marrow cells, macrophages, monocytes, dendritic cells, or Bcells) they could also be any of a wide range of types including,without limitation, fibroblasts, epithelial cells, endothelial cells,keratinocytes, or muscle cells in which they act as a source of theB7-H1 polypeptide or functional fragment for as long as they survive inthe subject. The use of hemopoietic cells, that include the above APC,would be particular advantageous in that such cells would be expected tohome to, among others, lymphoid tissue (e.g., lymph nodes or spleen) andthus the B7-H1 polypeptide or functional fragment would be produced inhigh concentration at the site where they exert their effect, i.e.,enhancement of an immune response. In addition, if APC are used, the APCexpressing the exogenous B7-H1 molecule can be the same APC thatpresents an alloantigen or antigenic peptide to the relevant T cell. TheB7-H1 can be secreted by the APC or expressed on its surface. Prior toreturning the recombinant APC to the patient, they can optionally beexposed to sources of antigens or antigenic peptides of interest, e.g.,those of tumors, infectious microorganisms, or autoantigens. The samegenetic constructs and trafficking sequences described for the in vivoapproach can be used for this ex vivo strategy. Furthermore, tumorcells, preferably obtained from a patient, can be transfected ortransformed by a vector encoding a B7-H1 polypeptide or functionalfragment thereof. The tumor cells, preferably treated with an agent(e.g., ionizing irradiation) that ablates their proliferative capacity,are then returned to the patient where, due to their expression of theexogenous B7-H1 (on their cell surface or by secretion), they canstimulate enhanced tumoricidal T cell immune responses. It is understoodthat the tumor cells which, after transfection or transformation, areinjected into the patient, can also have been originally obtained froman individual other than the patient.

The ex vivo methods include the steps of harvesting cells from asubject, culturing the cells, transducing them with an expressionvector, and maintaining the cells under conditions suitable forexpression of the B7-H1 polypeptide or functional fragment. Thesemethods are known in the art of molecular biology. The transduction stepis accomplished by any standard means used for ex vivo gene therapy,including calcium phosphate, lipofection, electroporation, viralinfection, and biolistic gene transfer. Alternatively, liposomes orpolymeric microparticles can be used. Cells that have been successfullytransduced are then selected, for example, for expression of the codingsequence or of a drug resistance gene. The cells may then be lethallyirradiated (if desired) and injected or implanted into the patient.

Methods of Screening for Compounds that Inhibit or Enhance ImmuneResponses.

The invention provides methods for testing compounds (small molecules ormacromolecules) that inhibit or enhance an immune response. Such amethod can involve, e.g., culturing a B7-H1 polypeptide of the invention(or a functional fragment thereof) with T cells in the presence of a Tcell stimulus (see above). Useful B7-H1 polypeptides include those withamino acid sequences identical to wild-type sequences or they cancontain one or more (e.g., one, two, three, four, five, six, seven,eight, nine, 10, 12, 14, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100or more) conservative substitutions. The B7-H1 polypeptide can be insolution or membrane bound (e.g., expressed on the surface of the Tcells) and it can be natural or recombinant. Compounds that inhibit theT cell response will likely be compounds that inhibit an immune responsewhile those that enhance the T cell response will likely be compoundsthat enhance an immune response.

The invention also relates to using B7-H1 or functional fragmentsthereof to screen for immunomodulatory compounds that can interact withB7-H1. One of skill in the art would know how to use standard molecularmodeling or other techniques to identify small molecules that would bindto T cell interactive sites of B7-H1. One such example is provided inBroughton (1997) Curr. Opin. Chem. Biol. 1, 392-398.

A candidate compound whose presence requires at least 1.5-fold (e.g.,2-fold, 4-fold, 6-fold, 10-fold, 150-fold, 1000-fold, 10,000-fold, or100,000-fold) more B7-H1 in order to achieve a defined arbitrary levelof T cell activation than in the absence of the compound can be usefulfor inhibiting an immune response. On the other hand, a candidatecompound whose presence requires at least 1.5 fold (e.g., 2-fold,4-fold, 6-fold, 10-fold, 100-fold, 1000-fold, 10,000 fold, or100,000-fold) less B7-H1 to achieve a defined arbitrary level of T cellactivation than in the absence of the compound can be useful forenhancing an immune response. Compounds capable of interfering with ormodulating B7-H1 function are good candidates for immunosuppressiveimmunoregulatory agents, e.g., to modulate an autoimmune response orsuppress allogeneic or xenogeneic graft rejection.

B7-H1 Antibodies

The invention features antibodies that bind to either or both of theB7-H1 polypeptides or fragments of such polypeptides. Such antibodiescan be polyclonal antibodies present in the serum or plasma of animals(e.g., mice, rabbits, rats, guinea pigs, sheep, horses, goats, cows, orpigs) which have been immunized with the relevant B7-H1 polypeptide orpeptide fragment using methods, and optionally adjuvants, known in theart. Such polyclonal antibodies can be isolated from serum or plasma bymethods known in the art. Monoclonal antibodies that bind to the abovepolypeptides or fragments are also embodied by the invention. Methods ofmaking and screening monoclonal antibodies are well known in the art.

Once the desired antibody-producing hybridoma has been selected andcloned, the resultant antibody can be produced in a number of methodsknown in the art. For example, the hybridoma can be cultured in vitro ina suitable medium for a suitable length of time, followed by therecovery of the desired antibody from the supernatant. The length oftime and medium are known or can be readily determined.

Additionally, recombinant antibodies specific for B7-H1, such aschimeric and humanized monoclonal antibodies comprising both human andnon-human portions, are within the scope of the invention. Such chimericand human monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example, using methods described inRobinson et al., International Patent Publication PCT/US86/02269; Akiraet al., European Patent Application 184,187; Taniguchi, European PatentApplication 171,496; Morrison et al., European Patent Application173,494; Neuberger et al. PCT Application WO 86/01533; Cabilly et al.,U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application125,023; Better et al. (1988) Science 240, 1041-43; Liu et al. (1987) J.Immunol. 139, 3521-26; Sun et al. (1987) PNAS 84, 214-18; Nishimura etal. (1987) Canc. Res. 47, 999-1005; Wood et al. (1985) Nature 314,446-49; Shaw et al. (1988) J. Natl. Cancer Inst. 80, 1553-59; Morrison,(1985) Science 229, 1202-07; Oi et al. (1986) BioTechniques 4, 214;Winter, U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321, 552-25;Veroeyan et al. (1988) Science 239, 1534; and Beidler et al. (1988) J.Immunol. 141, 4053-60.

Also included within the scope of the invention are antibody fragmentsand derivatives which contain at least the functional portion of theantigen binding domain of an antibody that binds specifically to B7-H1.Antibody fragments that contain the binding domain of the molecule canbe generated by known techniques. For example, such fragments include,but are not limited to: F(ab′)₂ fragments which can be produced bypepsin digestion of antibody molecules; Fab fragments which can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments; andFab fragments which can be generated by treating antibody molecules withpapain and a reducing agent. See, e.g., National Institutes of Health, 1Current Protocols In Immunology, Coligan et al., ed. 2.8, 2.10 (WileyInterscience, 1991). Antibody fragments also include Fv (e.g., singlechain Fv (scFv)) fragments, i.e., antibody products in which there areno constant region amino acid residues. Such fragments can be producedfor example, as described in U.S. Pat. No. 4,642,334 which isincorporated herein by reference in its entirety.

The following examples are meant to illustrate, not limit, theinvention.

EXAMPLE 1 Materials and Methods

Cloning of hB7-H1 cDNA and construction of Ig fusion proteins. The 5′and 3′ ends of hB7-H1 cDNA were amplified by PCR from a human placentacDNA library synthesized by SMART PCR cDNA synthesis kit (Clontech, PaloAlto, Calif.). The primer pairs used for the PCR were derived from theplacenta library plasmid and from the expressed sequence tag (EST) cloneAA292201. A cDNA clone that included an orf encoding hB7-H1 of hB7-H1cDNA was amplified by PCR from the same cDNA library by specific primersand cloned into the pcDNA3 vector (Invitrogen, Carlsbad, Calif.) andsequenced. The amino acid sequences of hB7-H1, B7-1 and B7-2 wereanalyzed using the ClustalW algorithm with BLOSUM 30 matrix (MacVector,Oxford Molecular Group). The hB7-H1Ig fusion protein was prepared byfusing the extracellular domain of hB7H-1 to the CH2-CH3 domain of mouseIgG2a in the expression plasmid pmIgV and the resulting construct wastransfected into CHO cells. An analogous method was also used forpreparation of B7-1Ig, CTLA4Ig and ICOSIg fusion proteins. The fusionproteins were purified from culture supernatants by passage over aProtein G-Sepharose affinity columns (Pharmacia, Uppsala, Sweden) andthe purified fusion proteins were dialyzed into endotoxin-free PBS.

DNA transfection. Plasmids containing nucleic acid sequences encodingfull length hB7-H1 (pcDNA3-hB7-H1), B7-1 (pCDM8-B7.1) or controlparental vectors without coding sequences were transfected into 293cells or COS cells by calcium phosphate or DEAE-Dextran transfection(Promega, Madison, Wis.). After 48 hours of incubation, the expressionlevels of hB7-H1 or B7-1 on transfectants were determined byfluorescence flow cytometry (FFC) with an antiserum specific for hB7-H1or anti-B7-1 monoclonal antibody (mAb) (PharMingen), respectively.

Mice and cell lines. Female C57BL/6 (B6), DBA/2, and BALB/c mice werepurchased from the National Cancer Institute (Frederick, Md.).CD28^(−/−) mice with a B6 genetic background were kindly provided by Dr.Moses Rodrigues (Department of Immunology, Mayo Clinic, Rochester,Minn.). P815 mastocytoma, L1210 lymphoma, EL4 mouse T-cell lymphoma and293 human kidney epithelial cells were purchased from the American TypeCulture, Collection (Manassas, Va.). Cell lines were maintained in acomplete medium containing RPMI-1640 (Life Technologies, Rockville, Md.)supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, Utah),25 mM HEPES, penicillin G (100 U/ml) and streptomycin sulfate (100μg/ml).

T-cell and cytokine assays. For human T cell studies, PBMC were isolatedfrom the blood of healthy human volunteer donors by Ficoll-Hypaquegradient centrifugation. The PBMC were passed through a nylon woolcolumn to obtain purified T cells (˜85% of CD3⁺ cells), or weresubjected to further purification (>95% of CD3⁺ cells) using ananti-CD4/8 MACS magnetic bead system (Miltenyl Biotec, Germany). Forco-stimulation assays, purified T cells at a concentration of 1×10⁵cells/well were cultured in triplicate in 96-well flat-bottomedmicrotiter tissue culture plates that were pre-coated overnight withantibody specific for human CD3 (HITa3, PharMingen, Palo Alto, Calif.)and either hB7-H1Ig (5 μg/ml) or control Ig (purified mouse IgG2a ormurine 4-1BBIg fusion protein). In some experiments, the microtiterwells were coated with only antibody specific for CD3 and B7-1- orhB7-H1-transfected COS cells were used (10⁴ cell/well) as a source ofthe co-stimulatory molecules. To measure cytokine production,supernatants were collected at 24, 48 and 72 hours after initiation ofthe cultures and the concentrations of IL-2, IL-4, IN-γ and IL-10 weredetermined by sandwich ELISA (PharMingen) according to themanufacturer's instructions. Wells containing B7-1Ig or antibodyspecific for human CD28 (CD28.2, PharMingen) were included forcomparison or as a positive control, respectively. T cell proliferationwas determined by the addition of 1.0 μCi [³H]-thymidine per well on day2 followed by at least 18 hours of additional culture. Incorporated[³H]-thymidine was determined using a MicroBeta TriLux liquidscintillation counter (Wallac, Finland).

For mixed lymphocyte reaction (MLR) assays, purified human T cells(2×10⁵ cells/well) were co-cultured in triplicate with allogeneic humanPBMC (4000 Rad-irradiated) at 2×10⁵ cells/well in the presence ofsoluble hB7-H1Ig or control Ig. Four days later, T cell proliferationwas determined by [³H]-thymidine incorporation. Neutralizing mAbspecific for human IL-2 (Clone MQ1-17H12, PharMingen) was added at 8μg/ml in the beginning of T cell cultures. Polymyxin B (10 μg/ml) wasalso included in the assays of cell proliferation and cytokine secretionto completely neutralize any contaminating endotoxin.

For mouse T cell studies, T cells were purified by passing lymph node orspleen cells trough a nylon wool column. CD4+ or CD8+ T cells werepositively selected by magnetic sorting using FITC-conjugated mAbagainst CD4 or CD8 and microbeads coated with antibody specific forfluorescein isothiocyanate (FITC) (MiltenyiBiotec, Auburn, Calif.)according to the manufacture's instructions. The purity of isolated CD4⁺and CD8⁺ T cells was >95% by FFC with mAb specific for mouse CD4 andCD8, respectively. Purified T cells at 2×10⁶/ml from mouse spleens werecultured in 96-well plates that were pre-coated with mAb specific formouse CD3 in the presence of mB7-H1Ig or control mouse IgG2a (“controlIg”) also coated onto the culture well bottoms. mAb specific for mouseCD28 (2.5 μg/ml) was used in soluble form as a positive controlco-stimulator. Proliferation of T cells was determined by incorporationof [³H]-thymidine (1.0 μCi/well) added 15 h before harvesting of the3-day cultures. [³H]-thymidine incorporation was determined by aMicroBeta TriLux liquid scintillation counter (Wallac, Turku, Finland).To detect cytokines, supernatants were collected between 18-72 h ofculture and the concentrations of IFN-γ, IL-2, IL-10, IL-4, and GM-CSFwere measured by sandwich ELISA following the manufacturer's(PharMingen) instructions.

Nucleic acid analysis. Northern blot analysis of human RNA was carriedout using commercially available human multiple tissue Northern blotmembranes (Clontech, Palo Alto, Calif.). Membranes were incubated inExpressHyb hybridization solution (Clontech) for 30 min at 68° C. Therandom-primed cDNA probe was full length hB7-H1 encoding cDNA (870 bp)labeled using [³²P]-dCTP. A ³²P-labeled human β-actin cDNA probe (2.0kb) was used as a control. Hybridization was carried out for 1 hr at 68°C., the membranes were washed 3 times in 2×SSC containing 0.05% SDS, andwere then exposed at −80° C. to x-ray film.

Tissue distribution of mB7-H1 mRNA was carried out using commerciallyavailable multiple tissue mouse RNA dot blot membranes (Clontech)according to the manufacturer's instructions. The random-primed cDNAprobe was full-length mB7-H1 encoding cDNA and was labeled using[³²P]-dCTP. The hybridization was performed for 16 h at 65° C. Afterwashing four times with 2×SSC containing 0.05% SDS, the membranes wereexposed at −80° C. to x-ray films.

Antibodies and fusion proteins. Rabbit antibodies against mB7-H1 proteinwere prepared by Cocalico Biologicals (Reamstown, Pa.) by immunizationof rabbits with a keyhole limpet hemocyanin (KLH)-conjugated hydrophilicpeptide spanning amino acids 95-119 of mB7-H1 (“peptide 95-119”)(GNAALQITDVKLQDAGVYCCIISYG) (SEQ ID NO:16). Polyclonal antibody waspurified from rabbit serum using an affinity column containing insolublematrix material conjugated with the peptide 95-119. Both ELISA and FFCanalysis of COS cells transfected with an expression vector containingcDNA encoding mB7-H1 demonstrated that the polyclonal antibody boundspecifically to mB7-H1. Purified mAb specific for mouse CD3 and mouseCD28 and FITC-conjugated mAb specific for mouse CD4, mouse CD8, andmouse CD40L, phycoerythrin (PE)-conjugated mAb specific for mouse CD3,mouse B220 and mouse Mac-1 were purchased from PharMingen (SanDiego,Calif.). FITC-conjugated goat antibody specific for rabbit IgG waspurchased from Southern Biotechnology Associates (Birmingham, Ala.).Purified rabbit IgG and hamster 190 were purchased from Rockland(Gilbertsville, Pa.).

To prepare the mB7-H1Ig fusion protein, cDNA encoding the mB7-H1extracellular domain was generated by RT-PCR using the sense primer5′-CAGGAATTCACCATGAGGATATTTGCTG-3′ (SEQ ID NO:17) and the anti-senseprimer 5′-CATCAGATCTATGTGAGTCCTGTTCTGTG-3′ (SEQ ID NO:18) from mouse Tcell mRNA. After digestion with EcoRI and BglII, the PCR products werefused to the CH2-CH3 domain of mouse IgG2a heavy chain in the expressionplasmid pmIgV [Dong et al. (1999) Nature Med 5, 1365-1369]. Theresulting plasmid, pmB7-H1Ig, was transfected into CHO cells. Stablytransfected cells were cultured in serum-free CHO media (LifeTechnologies). The mB7-H1Ig in the supernatants was purified using aprotein O-Sepharose column (Pierce, Rockford, Ill.) and dialyzed intoLPS-free PBS. The endotoxin concentration was less than 1 pg/mg ofpurified protein according to the limulus amebocyte lysate assays (CAPECOD, Woods Hoke, Mass.). The mB7-1Ig fusion protein containing the extracellular domain of mB7-1 fused to the Ch2-CH3 domain of mouse IgG-2aheavy chain was prepared by an analogous method.

Fluorescence flow cytometry analysis. To prepare an antiserum specificfor hB7-H1, mice were immunized with purified hB7-H1Ig emulsified incomplete Freund's adjuvant (Sigma) and boosted three times with hB7-H1Igin incomplete Freund's adjuvant. Serum was collected and the specificitywas determined by ELISA and by FACS staining (1:1000 dilution) of hB7-H1cDNA-transfected 293 cells or COS cells. Pre-injection mouse serum wasused as a control.

To prepare activated human T and B cells, freshly isolated human PBMC(10×10⁶ cells/ml) were activated with 5 μg/ml of PHA (Sigma) or 10 μg/mlof LPS (Sigma), respectively. For preparation of activated monocytes,adherent PBMCs were cultured in 1,500 IU/ml of recombinant human IFN-γ(Biosource, Camarillo, Calif.) and 100 ng/ml of LPS. All cultures wereharvested and analyzed at 48 hours. For direct immunofluorescencestaining, T cells were incubated at 4° C. with 1 μg of eitherfluorescein-(FITC) or phycoerythrin-(PE) conjugated mAb for 30 min andanalyzed by FACScan flow cytometry (Becton Dickinson, Mountain View,Calif.) with Cell Quest software (Becton Dickinson) as describedpreviously. The mAb specific for CD3 (UCHT1), CD4 (RPA-T4), CD8(RPA-T8), CD14 (M5E2), CD19 (B43), CD28 (CD28.2), CD80 (B1) werepurchased from PharMingen. For indirect immunofluorescence staining,cells were first incubated with anti-hB7-H1 antibody (1:1000), 5 μg ofICOSIg or CTLA4Ig at 4° C. After 30 min, the cells were washed andfurther incubated with FITC- (Biosource, Camarillo, Calif.) orPE-conjugated (Southern Biotechnology Associates, Inc., Birmingham,Ala.) goat anti-human or anti-mouse IgG F(ab′)₂ for 30 min at 4° C. Thehuman or mouse IgG1 protein (Sigma) or mouse 4-1BBIg (mouse 4-1BBextracellular domain fused with the Fc of human IgG1 or mouse IgG2a) wasused as control Ig. In some experiments, Fc receptors were blocked byhuman or mouse Ig before incubation with FITC- or PE-conjugated mAbs.

For indirect immunofluorescence analysis of mouse cells, the cells wereincubated with the antibodies at 4° C. for 30 min in the presence ofblocking mAb specific for CD16/32 (Fc receptor) Pharmingen). The cellswere washed and further incubated with FITC-conjugated anti-rabbit IgG.The cells were then stained with PE-conjugated mAb specific for mouseCD3, mouse B220, or mouse Mac-1. Fluorescence was analyzed with a FACSCalibur flow cytometer and analyzed with Cell Quest software (BectonDickinson, Mountain View, Calif.). To prepare activated mouse T cells,nylon-wool-purified mouse T cells (>75% CD3⁺ cells) at a concentrationof 2×10⁶/ml were cultured with mAb specific for mouse CD28 (5 μg/ml) andmouse CD3 (5 μg/ml). For preparation of activated mouse B cells, mousesplenocytes were cultured with LPS (10 μg/ml; Sigma, St. Louis, Mo.).Mouse macrophages were obtained from the peritoneal cavities of micewhich had been injected with thioglycollate 7 days before. Foractivation, the mouse peritoneal exudate cells (PEC) were cultured withIFN-γ (10 U/ml) and LPS (100 ng/ml). All cultures were harvested and thecells analyzed at 48 h. To detect CD40L expression, CD4⁺ T cells werepurified by magnetic sorting (see above), cultured as indicated, andincubated with FITC-conjugated mAb to CD40L.

Cytotoxic T-lymphocyte (CTL) generation. To generatealloantigen-specific CTL activity in vitro, nylon wool purified T cells(2.5×10⁶/ml) from B6 splenocytes were stimulated in 24-well tissueculture plates with irradiated (10,000 rads) mock.P815, mB7-1⁺ P815, orB7-H1⁺ P815 cells (2.5×10⁵/ml) for 5 days. After the 5-day stimulation,CTL activities against P815 (H-2^(d)) and EL4 (H-2^(b)) were measured ina standard ⁵¹Cr release assay [Chen et al. (1994) J. Exp. Med 179,523-532; Li et al. (1996) J. Exp. Med. 183, 639-644].

To generate tumor-specific CTL activity in vivo, DBA/2 mice wereinoculated subcutaneously (s.c.) with 1×10⁶ mock.P815, mB7-1⁺ P815, ormB7-H1⁺ P815 cells. The draining lymph nodes were removed 7-10 d aftertumor injection and the suspended lymph node cells (3×10⁶/ml) werere-stimulated in 24 well tissue culture plates with wild type irradiated(10,000 rads) P815 cells (3×10⁵/ml) for 5 days. The cells were harvestedand their CTL activity was measured in a standard ⁵¹Cr release assayagainst wild type P815 tumor target cells.

In vivo induction and assay of TNP-specific antibody. Trinitrophenol(TNP) conjugated to KLH (TNP-KLH; 100 μg/mouse) (Biosearch Technologies,Novato, Calif.) in phosphate buffered saline (PBS) was injected i.p.into B6 mice on day 0. On days 1 and 4, the mice were injected i.p. with100 μg of control Ig, mB7-1Ig, or mB7-H1Ig. Sera were collected on days7 and 14. To measure TNP-specific antibodies in the sera, 0.3 mg/mlTNP-BSA (Biosearch Technologies) was coated onto the well-bottoms of96-well ELISA plates overnight at 4° C. Non-specific binding sites inthe ELISA plates were blocked with 10% FBS in PBS for 90 min at roomtemperature. After extensive washing, samples (diluted by 1/200-1/2000with PBS) were added and incubated for 2 h. The plates were then washedand biotinylated rat antibodies specific for mouse IgM, IgG1, IgG2a,IgG2b, or IgG3 (PharMingen) were added to the wells. The plates werefurther incubated for 1 h at room temperature. After washing the plates,horseradish peroxidase (HRP)-conjugated streptavidin (CaltagLaboratories, Burlingame, Calif.) was added to the wells and the plateswere incubated for 1 h at room temperature. The plates were washed andthe solutions in all wells was measured. 3,3′,5,5′-tetramethyl-benzidinesubstrate (Sigma) was added to the wells. The OD₄₅₀ for the solutions inall wells was measured.

T cell proliferation in response to KLH. B6 mice were immunized with 100μg TNP-KLH in IFA s.c or in PBS i.p. on day 0 and were injected i.p.with 100 μg of either mB7-H1Ig or control Ig on days 1 and 4. To detectT cell responses to KLH, draining lymph nodes and spleens were removedfrom immunized mice on day 7 and 14, respectively. Suspended lymph nodeor spleen cells were cultured with KLH at 1.56-100 μg/ml as indicated. Tcell proliferation in response to KLH was determined by addition of 1μCi/well [³H]-thymidine 15 h before harvesting of the 3-day cultures,[³H]-thymidine incorporation was measured with a MicroBeta TriLux liquidscintillation counter (Wallac).

EXAMPLE 2 Molecular Cloning and Expression Pattern of the hB7-H1 Gene

A homology search of the human cDNA EST database using published humanB7-1 and B7-2 amino acid sequences revealed an EST sequence (GeneBank#AA292201) encoding a homologue to human B7-1 and B7-2 molecules. The5′- and 3′-sequences were obtained by several independent reversetranscriptase-coupled polymerase chain reactions (RT-PCR) from a humanplacenta cDNA library utilizing vector and EST sequences as primers. A3,616 bp fragment that included the hB7-H1 encoding orf was cloned andsequenced (SEQ ID NO:5) (FIG. 1). The coding sequence for hB7-H1 (SEQ IDNO:2) spans nucleotides 72-951 of SEQ ID NO:5. The amino acid sequenceof full-length hB7-H1 (SEQ ID NO:1) is shown in FIG. 2 a. Theextracellular domain of hB7-H1 has greater homology to B7-1 (20% aminoacid identity) than to B7-2 (15%) (FIG. 2 b) whereas its cytoplasmicdomain is highly divergent from that of B7-1 and B7-2 based on analysisusing the McVector 6.5 software. The open reading frame of the geneencodes a type I transmembrane protein of 290 amino acids consisting ofa 22 amino acid signal peptide, Ig V-like domain, and Ig C-like domains,a hydrophobic transmembrane domain and a cytoplasmic tail of 30 aminoacids (FIG. 2 a). Four structural cysteines (labeled by stars in FIG. 2b), which are apparently involved in forming the disulfide bonds of theIg V and Ig C domains are well conserved in all B7 members (FIG. 2 b)[Fargeas, C. A. et al. (1995) J. Exp. Med. 182, 667-675; Bajorath, J. etal. (1994) Protein Sci. 3, 2148-50; Linsley, P. S. et al. (1994)Immunity 1, 793-801; Inaba, K et al. (1994) J. Exp. Med. 180, 1849-60;Freeman, G. J. et al. (1995) Immunity 2, 523-532]. In addition, thetyrosine residue in B7-1 (at position 87) and in B7-2 (at position 82)of the Ig V-like domain is conserved in hB7-H1 (at position 81) (FIG. 2b).

Northern blot analysis revealed that expression of the hB7-H1 mRNA wasabundant in heart, skeletal muscle, placenta and lung but was weak inthymus, spleen, kidney and liver (FIG. 3). The hB7-H1 mRNA was notdetectable in brain, colon, small intestine and peripheral bloodmononuclear cells (PBMC). In most of the tissues in which hB7-H1 mRNAwas detectable, two transcripts of approximately 4.1 and 7.2 kb werefound.

An expression plasmid containing the extracellular domain of hB7-H1fused in frame with the Fc portion (CH2 and CH3-domains) of the mouseIgG2a was constructed. The resulting product, hB7-H1Ig fusion protein,was purified from the supernatants of CHO cells transfected with theplasmid and was used for immunization of the mice to prepare ahB7-H1-specific antiserum. Fluorescence flow cytometry analysis usingthe hB7-H1-specific antiserum showed that freshly isolated CD3+ T andCD19+ B cells express negligible levels of hB7-H1 while a fraction(˜16%) of CD14+ monocytes constitutively express hB7-H1. hB7-H1 can,however, be up-regulated by cell activation. Approximately 30% ofPHA-treated CD3+ T cells and 90% of CD14+ monocytes (treated with IFN-γand LPS) express hB7-H1. Only 6% of CD19+ B cells after LPS activationexpress hB7-H1 (FIG. 4). Confirmatory results were obtained by RT-PCRanalysis.

Transfection of the plasmid pcDNA3-hB7-H1 into 293 cells (B7-H1/293cells) led to the expression of hB7-H1 as detected by hB7-H1-specificantiserum (FIG. 5 a). The binding of antibody was eliminated by theinclusion of soluble hB7-H1Ig in the staining mixture (FIG. 5 a, arrow),thereby demonstrating specificity of the antiserum. Neither CTLA4Ig norICOSIg bound to hB7-H1/293 cells. Although both CTLA4Ig and ICOSIg boundto Raji cells, the binding was not blocked by the inclusion of hB7-H1Ig(FIG. 5 a, arrows). Taken together with the observation that hB7-H1Igdid not bind to Jurkat cells (FIG. 5 b, right panel), despite theirconstitutive expression of CD28 (FIG. 5 b, left panel), the aboveresults indicate that hB7-H1 is not a ligand for CD28, CTLA-4, or ICOS.

EXAMPLE 3 Co-Stimulation of T Cell Proliferation hB7-H1

To assess whether hB7-H1 co-stimulates T-cell growth, T cells purified(>95% purity) from PBMC of healthy human donors were stimulated withhB7-H1Ig in the presence of suboptimal doses of mAb specific for humanCD3. T cell proliferation in 3-day cultures was determined byincorporation of [³H]-thymidine. hB7-H1Ig, immobilized on cultureplates, enhanced T cell proliferation up to 10-fold compared to thecontrol Ig in the presence of 5-20 ng/ml of mAb specific for human CD3,also immobilized on the culture plates. In the absence of mAb specificfor human CD3, hB7-H1Ig at a concentration up to 5 μg/ml induced no Tcell proliferation (FIG. 6 a). If hB7-H1Ig was included in the cultureswithout immobilization, its co-stimulatory effect was significantlydecreased. Consistent with this observation, the inclusion of solublehB7-H1Ig at levels of 0.6-5 μg/ml in allogeneic MLR moderately (˜2-fold)increased the proliferation of T cells (FIG. 6 b). Thus, hB7-H1 canpromote and co-stimulate proliferative responses of T, cells topolyclonal T cell stimuli and to allogeneic antigens.

EXAMPLE 4 hB7-H1 Co-Stimulation Preferentially Induces the Production ofIL-10 and the Co-Stimulatory Effect Requires IL-2

The levels of IL-2, IL-4, IFN-γ, and IL-10 produced by T cells afterco-stimulation with hB7-H1Ig, B7-1Ig, or mAb specific for human CD28 inthe presence of mAb specific for human CD3 (FIG. 7 a-7 d) were measured.Similar to B7-1Ig and anti-CD28, immobilized hB7-H1Ig antibodydramatically increased the production of IL-10 by T cells in response toimmobilized mAb specific for human CD3 after stimulation for 48 and 72hours (FIG. 7 a). IL-10 was not detected if T cells were co-stimulatedwith immobilized control Ig. The level of IFN-γ was also significantlyelevated by co-stimulation with immobilized hB7-H1Ig (FIG. 7 b). Incontrast to B7-1Ig and mAb specific for human CD28, hB7-H1Igco-stimulated low or negligible levels of IL-2 (FIG. 7 c) and IL-4 (FIG.7 d), respectively. These observations were reproducible in sixindependent experiments. These results show that co-stimulation byhB7-H1 preferentially stimulates the production of IL-10.

The production of IL-2, although low, peaked at 24 hours upon hB7-H1co-stimulation (FIG. 7 c), while IL-10 secretion started to increaseonly after 48 and 72 hours (FIG. 7 a). Increasing concentrations ofhB7-H1Ig led to a small increase (<1 ng/ml) of IL-2 secretion (FIG. 7e). To determine the roles of the early-produced IL-2, the effects ofmAb specific for human IL-2 on T cell proliferation and IL-10 productionin B7H-mediated Co stimulation were tested. Similar to T cellproliferation induced by B7-1-COS cells and immobilized mAb specific forhuman CD3, T cell proliferation induced by hB7-H1-COS cells and mAbspecific for human CD3 was blocked by inclusion of mAb specific forhuman IL-2 (FIG. 8 a). Furthermore, IL-10 secretion fromhB7-H1Ig-co-stimulated T cells was also inhibited by mAb specific forhuman IL-2 (FIG. 8 b). Therefore, the hB7-H1 co-stimulation of both Tcell growth and IL-10 secretion is an IL-2-dependent process.

EXAMPLE 5 hB7-H1 Co-Stimulation Increases as Apoptosis of Activated TCells

To determine the effect of hB7-H1 ligation on the viability of activatedT cells, the proportion of live T cells remaining after activation withan optimally activating dose of mAb specific for human CD3 in thepresence of immobilized hB7-H1Ig was determined by trypan blue staining.A consistent decrease of alive T cells was observed. At the end ofculture, T cells were stained with annexin V and propidium iodide (PI)to distinguish the early phase and late phase of apoptosis,respectively. The apoptotic cells in early phase (annexin V-positive,PI-negative) were significantly increased to 24.8% in the presence ofhB7-H1Ig compared to 14.2% in the absence of hB7-H1Ig in 5 experiments(P=0.001). A representative experiment is shown in FIG. 9 a (upperpanel). Similar results were obtained using hB7-H1Ig-treated Jurkatcells (control Ig: 38.3% vs. hB7-H1Ig: 54.6%) (FIG. 9 a, lower panel).The increased apoptosis was associated with upregulation of Fas and FasLexpression on hB7-H1 co-stimulated T cells (FIG. 9 b). These resultsindicated that hB7-H1 co-stimulation increased activation-induced T cellapoptosis moderately, and the increased apoptosis was associated withelevated expression of Fas and FasL.

EXAMPLE 6 Production of Monoclonal Antibodies Specific for hB7-H1

Using standard protocols, BALB/c mice were immunized with purifiedhB7-H1Ig and splenocytes from the immunized mice were fused withX63-AG8.653 mouse myeloma cells. Five hybridoma lines were found tosecrete antibodies specific for hB7-H1 in that, as detected byfluorescence flow cytometry, culture supernatants from these hybridomalines positively stained hB7-H1/293 cells but did not stain controlvector/293 cells. Furthermore, some of the antibodies inhibited theco-stimulatory activity of hB7-H1.

EXAMPLE 7 Molecular Cloning and Expression Pattern of a Mouse B7-H1(mB7-H1) Gene

Starting with two overlapping mouse EST clones (AA823166 and AA896104),and using a strategy similar to that for the hB7-H1 gene, a cDNAfragment that included an orf encoding mB7-H1 was cloned. The codingsequence for mB7-H1 (SEQ ID NO:4) (FIG. 10) was obtained and the aminoacid sequence of mB7-H1 (SEQ ID NO: 3) (FIG. 11) was derived from it.The length of mB7-H1 is identical to that of hB7-H1 and it has the sameconserved cysteine residues found in hB7-H1 (see Example 2). A cDNAfragment encoding full-length mB7-H1 was cloned into the pcDNA3 vector(Invitrogen, Carlsbad, Calif.) to give mB7-H1.pcDNA3.

mB7-H1, like hB7-H1, is a type I transmembrane protein of 290 aminoacids that has 69% overall amino acid homology to hB7-H1 (FIG. 12 a).Similar to other members of B7 family, mB7-H1 consists of an Ig V-likedomain, an Ig C-like domain, a hydrophobic transmembrane domain and acytoplasmic tail. mB7-H1 shares 20% homology to mouse B7-1, 14% to mouseB7-2, and 19% to mouse B7h/B7RP-1, based on analysis using McVector 6.5software (Clustal W Program) (FIG. 12 b).

RNA analysis revealed that mB7-H1 mRNA is abundant in mouse heart,spleen, lung, skeletal muscle and liver, and less abundant but presentin mouse kidney, liver, thymus, and thyroid. Thus, the expressionpattern of mB7-H1 mRNA is similar to that of human B7-H1 mRNA.Negligible expression of the mB7-H1 mRNA was observed in pancreas andtestis.

FFC analysis using the anti-mB7-H1 antibody showed that resting mouseCD3⁺ T cells do not express mB7-H1 (FIG. 13, upper panel). However, asmall fraction of B220⁺ mouse B cells and Mac-1⁺ mouse macrophagesexpressed a low level of mB7-H1 (FIG. 13, upper panel). Stimulation ofmouse T cells with antibodies specific for mouse CD3 and mouse CD28moderately increased the mB7-H1 expression on T cells. Activation ofmouse B cells with LPS and macrophages with LPS plus IFN-γ significantlyincreased the expression of mB7-H1 on their surfaces (FIG. 13, lowerpanel). Thus, mB7-H1, like hB7-H1, is an inducible cell surfacemolecule.

EXAMPLE 8 Co-Stimulation of Mouse T Cell Proliferation by mb7-H1

To investigate the costimulatory effect of mB7-H1, nylon-wool purifiedmouse T cells were activated with a suboptimal dose of mAb specific formouse CD3 (coated onto culture well bottoms at a concentration of 200ng/ml) and co-stimulated with various concentrations of mB7-H1Ig. inB7-H1Ig enhanced T cell proliferation by up to 5-fold compared tocontrol Ig (FIG. 14 a). The costimulatory effect of the mB7-H1Ig wasdose-dependent and dependent on the presence of mAb specific for mouseCD3 since in the absence of mAb specific for mouse CD3, mB7-H1Ig (up toa concentration of mB7-H1Ig 10 μg/ml) failed to stimulate theproliferation of T cells. When nylon-wool purified mouse T cells werecultured with 293 cells transfected with either mB7-H1.pcDNA3 or controlvector in the presence of suboptimal doses of mAb specific for mouseCD3, mB7-H1-transfected 293 cells also enhanced T cell proliferationsubstantially compared to the T cell proliferation in the presence ofthe control vector-transfected 293 cells. Thus, similar to hB7-H1,mB7-H1 costimulates T cell proliferation.

The role of CD28 in mB7-H1 costimulation was evaluated by comparing theeffects of mB7-H1Ig costimulation on T cells isolated from CD28^(−/−)mice and from normal mice. Nylon wool purified mouse T cells wereactivated with two suboptimal doses of antibody specific for mouse CD3(coated Onto the well bottoms of 96 well tissue culture plates at aconcentration of 0.125 μg/ml or 0.25 μg/ml) and either soluble antibodyspecific for mouse CD28 (2.5 μg/ml) or mB7-H1Ig or control Ig (bothcoated onto the well bottoms of 96 well tissue culture plates at aconcentration of 10 μg/ml). As shown in FIG. 14 b, while there was noco-stimulatory effect of anti-CD28 mAb on CD28^(−/−) T cells, mB7-H1Ig,induced the proliferation of both CD28^(−/−) (FIG. 14 b, right panel)and CD28^(+/+) (“wt”; FIG. 14 b, left panel) T cells to a similardegree. Therefore, mB7-H1 can costimulate T cell growth in aCD28-independent fashion.

In order to test whether mB7-H1 preferentially co-stimulates CD4⁺ orCD8⁺ T cells, purified CD4⁺ and CD8⁺ T cells were stimulated withmB7-H1Ig (same concentration as in the experiment shown in FIG. 14 b)and mAb specific for mouse CD3 (coated onto the well bottoms of 96 welltissue culture plates at a concentration of 200 ng/ml). Proliferation ofCD4⁺ T cells was enhanced about 10 fold by mB7-H1Ig and theproliferation of CD8⁺ T cells was only enhanced 2-3 fold mB7-H1Ig (FIG.14 c). Thus, the co-stimulatory effect of mB7-H1 is more potent on CD4⁺T cells than on CD8+ cells.

EXAMPLE 9 Co-Stimulation of Cytokine Production by mB7-H1

The levels of IL-10, IFN-γ, IL-2, IL-4 and GM-CSF produced by T cellsactivated with mAb specific for mouse CD3 and co-stimulated with eithermB7-H1Ig or anti-CD28 mAb were measured FIG. 15 a shows that mB7-H1Ig,similar to mAb specific for mouse CD28, co-stimulates the production ofhigh levels of IL-10 in the day 3 cultures. IL-10 was not detectable atday 3 when T cells were treated with either control Ig and mAb specificfor mouse CD3 or mAb specific for mouse CD3 alone. mB7-H1 and mAbspecific for mouse CD28 enhanced the production of IFN-γ and GM-CSF. Incontrast to mAb specific for mouse CD28, which induced high levels ofIL-2 and IL-4, mB7-H1Ig induced no or negligible levels of IL-2 and IL-4at all time points (FIG. 15 a). Thus, mB7-H1 and hB7-H1 co-stimulate theproduction of a similar spectrum of cytokines.

Since IL-2 was undetectable in culture supernatants from mB7-H1Igco-stimulated cultures, it seemed possible that mB7-H1 ligationinhibited IL-2 secretion. To test this possibility, the effect of mB7-H1on IL-2 secretion by T cells activated by mAb specific for mouse CD3 andco-stimulated with mAb specific for mouse CD28 was tested. FIG. 15 bshows that inclusion of immobilized mB7-H1Ig (at concentrations up to 10μg/ml) in the culture resulted in a small decrease in IL-2 productionduring the 18-48 h culture period that was statistically insignificant;in several repeat experiments no statistically significant decrease wasever seen. Similarly, mB7-H1Ig did not inhibit IL-2 production incultures in which T cells were activated by mAb specific for mouse CD3alone (FIG. 15 b). The results thus indicate that mB7-H1 ligation doesnot inhibit the production of IL-2.

EXAMPLE 10 Expression of mB7-H1 on Transfected P815 Tumor Cells andDecreased Growth Rate of the Transfected P815 Cells in Mice

Mouse (DBA/2) P815 mastocytoma cells were stably transfected with theexpression plasmid (mB7-H1.pcDNA3) containing the coding sequence formB7-H1 using FUGENE™ (Roche, Mannheim, Germany) according to themanufacturer's instructions. The transfected cells were selected incomplete medium containing G418 (1 mg/ml; Life Technologies) and weresubsequently cloned by limiting dilution. mB7-H1 expressing P815 cellswere identified by FFC using the above-described anti-mB7-H1 polyclonalantibody preparation. A representative clone (mB7-H1⁺P815) was selectedfor further studies. P815 clones transfected with the pc DNA vector(mock.P815) or MB7-1 (mB7-1⁺ P815) were generated similarly [Chen et al.(1994) J. Exp. Med. 179, 523-532]. Using a PE-conjugated rat polyclonalantibody specific for mB7-H1 (“anti-mB7H/PE”), mB7-H1 expression wasdetected by FFC on the mB7H-H1⁺ P815 cells (FIG. 18 b) but not on eithermock transfected P815 cells (“mock.P815”) (FIG. 16 b) or P815 cellstransfected with a construct encoding murine B7-1 (“mB7-1⁺ P815”) (FIG.17 b). On the other hand, the mB7-1⁺ P815 cells were stained with aFITC-conjugated mAb specific for murine B7-1 (“anti-mB7-1-FITC”) (FIG.17 a). Furthermore, inclusion of the mB7-H1 peptide used to make thepolyclonal anti-mB7-H1 antibody in the staining reaction mixturecompletely blocked binding of the polyclonal anti-mB7-H1 antibody to themB7-H1⁺ P815 cells.

Groups (5 mice per group) of DBA/2 mice were injected subcutaneously(s.c.) with either 2×10⁵ mock.P815 or mB7-H1⁺ P815 cells. The growthrate of the mock.P815 cells was significantly greater in 4 out of 5injected mice (FIG. 19 a) than in the 5 mice injected with mB7-H1⁺ P815(FIG. 19 b). These findings indicate that the mB7-H1⁺ P815 cells weresignificantly more immunogenic than mock.P815 cells and, therefore, thatexpression of mB7-H1 expression by P815 cells enhances their ability toelicit protective immunity.

EXAMPLE 11 mB7-H1 Costimulation Fails to Enhance Allogeneic andSyngeneic CTL Responses

To examine the effect of mB7-H1 on the generation of allogeneic CTL invitro, nylon wool-purified T cells from B6 mice (H-2^(b)) wereco-cultured with irradiated mock.P815 (H-2^(d)), mB7-1⁺ P815, or mB7-H1⁺P815 cells for 5 days and the CTL activity of cells harvested from thecultures was tested against wild-type P815 target cells in standard ⁵¹Crrelease assays at the indicated effector to target cell ratios (“E/Tratio”). As depicted in FIG. 20 a, mB7-H1⁺ P815 cells and mock.P815cells were poor stimulators of CTL activity. In contrast, mB7-1⁺ P815cells elicited strong P815-specific CTL activity. The CTL induced bymB7-1 were alloantigen-specific since they did not lyse responder (B6)H-2 haplotype (H-2^(b)) EL4 tumor target cells. Thus, mB7-H1 expressiondoes not facilitate the generation of allogeneic CTL.

The ability of mB7-H1⁺ P815 cells to stimulate P815 tumor specific CTLin vivo was tested. DBA/2 mice were injected s.c. with mock.P815, mB7-1⁺P815, or mB7-H1⁺ P815 cells. Tumor-draining lymph nodes were removed 7days later and T cells isolated from them were cultured with wild-typeP815 cells for 5 days. Cells harvested from the cultures were tested forCTL activity against wild-type P815 target cells in a standard ⁵¹Crrelease assay at the indicated E/T ratios (FIG. 20 b). Effector cellsfrom mice injected with mB7-H1⁺ P815 showed slightly increased CTLactivity against P815 cells compared to effector cells from themock.P815-injected mice; this difference in CTL activity was howeverstatistically insignificant. In contrast, mB7-1⁺ P815 cells elicitedstrong CTL activity. CTL activity was P815 tumor-specific sincesyngeneic L1210 tumor target cells were not lysed. Thus, expression ofmB7-H1 in P815 cells does not enhance the induction of CTL activityagainst P815 tumor antigens.

EXAMPLE 12 B7-H1 Costimulation Amplifies Antigen-Specific T Helper CellResponses T Cell-Dependent Humoral Responses, and Expression of CD40L onT Cells

To investigate the effect of mB7-H1 costimulation on T helper cellfunction, B6 mice were immunized with TNP-conjugated KLH on day 0, andwere injected with mB7-H1Ig at day 1 and day 4. The in vitroproliferative responses of T cells obtained from both lymph nodes andspleens of the immunized mice to various concentrations of KLH weremeasured. As shown in FIG. 21, T cells from both spleens and lymph nodesof TNP-KLH-immunized mice proliferated in response to KLH in a dosedependent fashion. Administration of mB7-H1Ig to TNP-KLH-immunized miceamplified the subsequent in vitro proliferative responses of T cells byup to 2-3 fold. These results indicate that mB7-H1 co-stimulationenhances T helper cell responses in vivo.

The effect of mB7-H1 co-stimulation on the generation ofantigen-specific antibodies to TNP was investigated in a system wellrecognized as measuring helper T cell-dependent antibody responses[Marrack and Kappler (1975) J. Immunol. 114, 1116-1125; Romano et al.(1975) Proc. Natl. Acad. Sci. USA 72, 4555-4558]. Thus, the level ofantibodies specific for TNP in the sera of mice immunized with TNP-KLHwas measured after treatment with control Ig, mB7-1Ig, or mB7-H1Ig. Inpreliminary experiments, a significant increase in the total anti-TNPIgG level was observed in sera of mice immunized with TNP-KLH andtreated with mB7-H1Ig compared to sera of mice immunized with TNP-KLHand treated with control mIg. The relative levels of IgM and individualIgG subclass (IgG1, IgG2a, IgG2b and IgG3) anti-TNP antibodies elicitedby TNP-KLH immunization and various co-stimulations were each measured.As shown in FIG. 22, the amount of TNP-specific IgG2a antibody wasincreased significantly in the sera of mice immunized with TNP-KLH andtreated with mB7-H1Ig. The effect was different from that elicited bymB7-1Ig in which the levels of antibodies specific for TNP of other IgGsubclasses (IgG1 and IgG2b) were also significantly increased (FIG. 22).Thus, mB7-H1 costimulation enhances T helper cell proliferation and Thelper-dependent antibody responses, particularly IgG2a responses. TheCD40-CD40 ligand (CD40L) interaction in T helper cell-B cellinteractions is critical for the generation of antibody responses andfor Ig class switching [Calderhead et al. (2000) Curr. Top. Microbiol.Immunol. 245, 73-99]. The effect of co-stimulation with mB7-H1Ig on thelevel of CD40L on T cells was investigated. Purified CD4+ T cells fromB6 mice were stimulated with a suboptimal concentration of mAb specificfor mouse CD3 in the presence of mB7-H1Ig or mAb specific for mouseCD28. Expression of CD40L on T cells was detected with a mAb specificfor mouse CD40L by FFC. mB7-H1Ig co-stimulation upregulated CD40Lrapidly (25.3% after 4 h incubation) compared to co-stimulation withcontrol IgG (6.6%) or antibody specific for mouse CD28 (10.5%) (FIG. 23a). The level of CD40L was also higher after 24 h on T cellsco-stimulated with mB7-H1Ig than on T cells co-stimulated with theeither control Ig or mB7-1Ig (FIG. 23 a). Similar results were obtainedwith using an optimal dose of mAb specific for mouse CD3 for activation(FIG. 23 b). Thus, triggering of the B7-H1 counter-receptor on T cellsrapidly upregulates the expression of CD40L.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

What is claimed is:
 1. An isolated antibody that binds specifically to aB7-H1 polypeptide which comprises (a) amino acids having the sequenceset forth in SEQ ID NO:1; or (b) amino acids that are encoded by apolynucleotide comprising nucleotides having the sequence set forth inSEQ ID NO:2.
 2. The isolated antibody of claim 1, which is a monoclonalantibody.
 3. The isolated antibody of claim 1, which binds to thepolypeptide comprising amino acids having the sequence set forth in SEQID NO:1.
 4. An antibody fragment that specifically binds to a B7-H1polypeptide which comprises (a) amino acids having the sequence setforth in SEQ ID NO:1; or (b) amino acids that are encoded by apolynucleotide comprising nucleotides having the sequence set forth inSEQ ID NO:2.
 5. The antibody fragment of claim 4, which is an F(ab)₂fragment, an Fab fragment or a single-chain Fv fragment (scFv) and whichspecifically binds to the polypeptide of (a) or (b).
 6. The monoclonalantibody of claim 2, which is a chimeric or a humanized monoclonalantibody.
 7. A hybridoma which produces a monoclonal antibody accordingto claim
 2. 8. The isolated antibody according to claim 1, which bindsto a polypeptide comprising amino acids having a sequence from aminoacid residue 23 to amino acid residue 290 of SEQ ID NO:1; or apolypeptide comprising one or more conservative substitutions in SEQ IDNO:1.
 9. The isolated antibody according to claim 1, which binds to apolypeptide comprising amino acids having a sequence from about aminoacid residue 20 to amino acid residue 290 of SEQ ID NO:1.
 10. Theisolated antibody according to claim 1, which binds to a polypeptidecomprising amino acids having a sequence from amino acid residue 23 toamino acid residue 290 of SEQ ID NO:1.
 11. An isolated antibody whichselectively binds to a B7-H1 polypeptide which (a) comprises amino acidshaving a sequence from amino acid residue 23 to amino acid residue 239of SEQ ID NO:1.
 12. An isolated antibody or an antigen binding fragmentthereof which selectively binds to the polypeptide of SEQ ID NO:1. 13.The isolated antibody according to claim 12 which is a polyclonal, amonoclonal, a chimeric, a human, a humanized antibody, or an antigenbinding antibody fragment thereof.
 14. The isolated antibody accordingto claim 12 which is an Fab antibody fragment, an F(ab′)2 antibodyfragment, a single chain antibody fragment, an scFv antibody fragment,or an antigen-binding fragment thereof.
 15. The isolated antibodyaccording to claim 12 which is attached to a solid substrate.
 16. Theisolated antibody of claim 12, which inhibits the co-stimulatoryactivity of said B7-H1 polypeptide of SEQ ID NO:1.
 17. The isolatedantibody of claim 12, which inhibits the co-stimulatory activity of saidB7-H1 polypeptide of SEQ ID NO:1 on a T-cell.
 18. An isolated cell thatis capable of expressing the antibody of claim
 1. 19. A compositioncomprising the antibody of claim 1 and a pharmaceutically acceptableexcipient.
 20. The composition of claim 19, wherein said antibody is apolyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanantibody, or a humanized antibody.
 21. The composition of claim 19,wherein said antibody inhibits the co-stimulatory activity of said B7-H1polypeptide of SEQ ID NO:1.
 22. A method for detecting the presence of aB7-H1 polypeptide in a sample comprising: (a) contacting the sample withthe isolated antibody of claim 1; and (b) determining the binding ofsaid antibody to the polypeptide in the sample to thereby detect thepresence of a B7-H1 polypeptide in the sample.
 23. An isolated antibodyor an antigen binding fragment thereof which specifically binds to apolypeptide consisting of amino acids having the sequence set forth inSEQ ID NO:1.
 24. A method of making an antibody, or an antigen bindingfragment thereof comprising: providing a polypeptide which is (a) apolypeptide comprising amino acids having the sequence set forth in SEQID NO:1; or (b) a polypeptide which is encoded by a polynucleotidecomprising nucleotides having the sequence set forth in SEQ ID NO:2 toan immunocompetent vertebrate; and harvesting from the vertebrate bloodor serum comprising an antibody which specifically binds to saidpolypeptide, or an antigen binding fragment thereof.
 25. A compositioncomprising the antibody fragment according to claim 4 and apharmaceutically acceptable excipient.
 26. The composition according toclaim 25, wherein said antibody fragment is an Fab fragment, an F(ab′)2fragment, a single chain antibody fragment, an scFv antibody fragment,or an antigen-binding fragment thereof.
 27. An antibody or an antigenbinding fragment thereof which binds to a soluble B7-H1 polypeptidewhich is not membrane-bound, wherein said soluble B7-H1 polypeptidecomprises amino acid domain(s) having the sequence(s) shown in FIG. 2A(SEQ ID NO:1) such that the polypeptide is soluble.